Early localization of NPA58, a rat nuclear pore-associated protein, to the reforming nuclear envelope during mitosis

We have studied the mitotic reassembly of the nuclear envelope, using antibodies to nuclear marker proteins and NPA58 in F-111 rat fibroblast cells. In earlier studies we have proposed that NPA58, a 58 kDa rat nuclear protein, is involved in nuclear protein import. In this report, NPA58 is shown to be localized on the cytoplasmic face of the envelope in interphase cells, in close association with nuclear pores. In mitotic cells NPA58 is dispersed in the cytoplasm till anaphase. The targeting of NPA58 to the reforming nuclear envelope in early telophase coincides with the recruitment of a well-characterized class of nuclear pore proteins recognized by the antibody mAb 414, and occurs prior to the incorporation of lamin B1 into the envelope. Significant protein import activity is detectable only after localization of NPA58 in the newly-formed envelope. The early targeting of NPA58 is consistent with its proposed role in nuclear transport.     

Stathmin inhibition enhances okadaic acid-induced mitotic arrest: a potential role for stathmin in mitotic exit.

Stathmin is a microtubule-destabilizing phosphoprotein that plays a critical role in the regulation of mitosis. The microtubule-depolymerizing activity of stathmin is lost upon phosphorylation in mitosis. Although the role of phosphorylation of stathmin by p34(cdc2) kinase in the assembly of the mitotic spindle is well established, the role of dephosphorylation of stathmin in mitosis is unknown. In this study, we tested the hypothesis that dephosphorylation of stathmin may be critically important for the depolymerization of the mitotic spindle and the exit from mitosis. We compared the effects of okadaic acid, a specific inhibitor of serine/threonine protein phosphatases, on different parameters of mitotic progression in the presence or absence of stathmin deficiency. Because okadaic acid prevents dephosphorylation of stathmin and results in accumulation of the inactive phosphorylated form, exposure to okadaic acid would be expected to have a more profound effect on mitosis in the presence of relative stathmin deficiency. We found that inhibition of stathmin expression results in increased sensitivity to the antimitotic effects of okadaic acid. This was reflected by increased growth inhibition associated with mitotic arrest. A vast majority of the stathmin-inhibited cells were found to be arrested in late metaphase/anaphase and had severe mitotic spindle abnormalities. Exposure to okadaic acid also resulted in a bigger ratio of polymerized/unpolymerized tubulin in stathmin-inhibited cells relative to control cells. Because the only difference between the control and the stathmin-inhibited cells is the deficiency of stathmin in the latter, the increased susceptibility of the stathmin-inhibited cells to okadaic acid-induced mitotic arrest implies a role for stathmin in the later stages of mitosis.     

The mitotic phosphorylation cycle of the cis-Golgi matrix protein GM130

The cis-Golgi matrix protein GM130 is phosphorylated in mitosis on serine 25. Phosphorylation inhibits binding to p115, a vesicle-tethering protein, and has been implicated as an important step in the mitotic Golgi fragmentation process. We have generated an antibody that specifically recognizes GM130 phosphorylated on serine 25, and used this antibody to study the temporal regulation of phosphorylation in vivo. GM130 is phosphorylated in prophase as the Golgi complex starts to break down, and remains phosphorylated during further breakdown and partitioning of the Golgi fragments in metaphase and anaphase. In telophase, GM130 is dephosphorylated as the Golgi fragments start to reassemble. The timing of phosphorylation and dephosphorylation correlates with the dissociation and reassociation of p115 with Golgi membranes. GM130 phosphorylation and p115 dissociation appear specific to mitosis, since they are not induced by several drugs that trigger nonmitotic Golgi fragmentation. The phosphatase responsible for dephosphorylation of mitotic GM130 was identified as PP2A. The active species was identified as heterotrimeric phosphatase containing the Balpha regulatory subunit, suggesting a role for this isoform in the reassembly of mitotic Golgi membranes at the end of mitosis.     

Dephosphorylation of Barrier-to-autointegration Factor by Protein Phosphatase 4 and Its Role in Cell Mitosis

Barrier-to-autointegration factor (BAF or BANF1) is highly conserved in multicellular eukaryotes and was first identified for its role in retroviral DNA integration. Homozygous BAF mutants are lethal and depletion of BAF results in defects in chromatin segregation during mitosis and subsequent nuclear envelope assembly. BAF exists both in phosphorylated and unphosphorylated forms with phosphorylation sites Thr-2, Thr-3, and Ser-4, near the N terminus. Vaccinia-related kinase 1 is the major kinase responsible for phosphorylation of BAF. We have identified the major phosphatase responsible for dephosphorylation of Ser-4 to be protein phosphatase 4 catalytic subunit. By examining the cellular distribution of phosphorylated BAF (pBAF) and total BAF (tBAF) through the cell cycle, we found that pBAF is associated with the core region of telophase chromosomes. Depletion of BAF or perturbing its phosphorylation state results not only in nuclear envelope defects, including mislocalization of LEM domain proteins and extensive invaginations into the nuclear interior, but also impaired cell cycle progression. This phenotype is strikingly similar to that seen in cells from patients with progeroid syndrome resulting from a point mutation in BAF.     

Identification of tyrosine-phosphorylated proteins associated with the nuclear envelope.

The nuclear envelope separates the nucleoplasm from the rest of the cell. Throughout the cell cycle, its structural integrity is controlled by reversible protein phosphorylation. Whereas its phosphorylation-dependent disassembly during mitosis is well characterized, little is known about phosphorylation events at this structure during interphase. The few characterized examples cover protein phosphorylation at serine and threonine residues, but not tyrosine phosphorylation at the nuclear envelope. Here, we demonstrate that tyrosine phosphorylation and dephosphorylation occur at the nuclear envelope of intact Neuro2a mouse neuroblastoma cells. Tyrosine kinase and phosphatase activities remain associated with purified nuclear envelopes. A similar pattern of tyrosine-phosphorylated nuclear envelope proteins suggests that the same tyrosine kinases act at the nuclear envelope of intact cells and at the purified nuclear envelope. We have also identified eight tyrosine-phosphorylated nuclear envelope proteins by 2D BAC/SDS/PAGE, immunoblotting with phosphotyrosine-specific antibodies, tryptic in-gel digestion, and MS analysis of tryptic peptides. These proteins are the lamina proteins lamin A, lamin B1, and lamin B2, the inner nuclear membrane protein LAP2beta, the heat shock protein hsc70, and the DNA/RNA-binding proteins PSF, hypothetical 16-kDa protein, and NonO, which copurify with the nuclear envelope.     

The dephosphorylated form of the anaphase-promoting complex protein Cdc27/Apc3 concentrates on kinetochores and chromosome arms in mitosis

Cell cycle regulated protein ubiquitination and degradation within subcellular domains may be essential for the normal progression of mitosis. Cdc27 is a conserved component of an essential M-phase ubiquitin-protein ligase called the anaphase-promoting complex/cyclosome. We examined the subcellular distribution of Cdc27 in greater detail in mammalian cells and found Cdc27 concentrated at spindle poles and on spindle microtubules as previously described, but also found Cdc27 at kinetochores and along chromosome arms. This localization was not dependent on intact microtubules. While the great majority of Cdc27 protein in M phase cells is highly phosphorylated, only the dephosphorylated form of Cdc27 was found associated with isolated chromosomes. Kinases that also associate with isolated chromosomes catalyzed the in vitro phosphorylation of the chromosome-associated Cdc27. Microinjection of anti-Cdc27 antibody into cells causes arrest at metaphase. Microinjection of cells with anti-Mad2 antibody normally induces premature anaphase onset resulting in catastrophic nondisjunction of the chromosomes. However, coinjection of anti-Cdc27 antibody with anti-Mad2 antibody resulted in metaphase arrest. The association of dephosphorylated APC/C components with mitotic chromosomes suggests mechanisms by which the spindle checkpoint may regulate APC/C activity at mitosis.     

Phosphorylation regulates the dynamic interaction of RCC1 with chromosomes during mitosis

The small GTPase Ran has multiple roles during the cell division cycle, including nuclear transport, mitotic spindle assembly, and nuclear envelope formation. However, regulation of Ran during cell division is poorly understood. Ran-GTP is generated by the guanine nucleotide exchange factor RCC1, the localization of which to chromosomes is necessary for the fidelity of mitosis in human cells. Using photobleaching techniques, we show that the chromosomal interaction of human RCC1 fused to green fluorescent protein (GFP) changes during progression through mitosis by being highly dynamic during metaphase and more stable toward the end of mitosis. The interaction of RCC1 with chromosomes involves the interface of RCC1 with Ran and requires an N-terminal region containing a nuclear localization signal. We show that this region contains sites phosphorylated by mitotic protein kinases. One site, serine 11, is targeted by CDK1/cyclin B and is phosphorylated in mitotic human cells. Phosphorylation of the N-terminal region of RCC1 inhibits its binding to importin alpha/beta and maintains the mobility of RCC1 during metaphase. This mechanism may be important for the localized generation of Ran-GTP on chromatin after nuclear envelope breakdown and may play a role in the coordination of progression through mitosis.     

Nuclear Localization of Cyclin B1 Controls Mitotic Entry After DNA Damage

Mitosis in human cells is initiated by the protein kinase Cdc2-cyclin B1, which is activated at the end of G2 by dephosphorylation of two inhibitory residues, Thr14 and Tyr15. The G2 arrest that occurs after DNA damage is due in part to stabilization of phosphorylation at these sites. We explored the possibility that entry into mitosis is also regulated by the subcellular location of Cdc2-cyclin B1, which is suddenly imported into the nucleus at the end of G2. We measured the timing of mitosis in HeLa cells expressing a constitutively nuclear cyclin B1 mutant. Parallel studies were performed with cells expressing Cdc2AF, a Cdc2 mutant that cannot be phosphorylated at inhibitory sites. Whereas nuclear cyclin B1 and Cdc2AF each had little effect under normal growth conditions, together they induced a striking premature mitotic phenotype. Nuclear targeting of cyclin B1 was particularly effective in cells arrested in G2 by DNA damage, where it greatly reduced the damage-induced G2 arrest. Expression of nuclear cyclin B1 and Cdc2AF also resulted in significant defects in the exit from mitosis. Thus, nuclear targeting of cyclin B1 and dephosphorylation of Cdc2 both contribute to the control of mitotic entry and exit in human cells.     

Threonine phosphorylation is associated with mitosis in HeLa cells

Phosphorylation and dephosphorylation of proteins play an important role in the regulation of mitosis and meiosis. In our previous studies we have described mitosis-specific monoclonal antibody MPM-2 that recognizes a family of phosphopeptides in mitotic cells but not in interphase cells. These peptides are synthesized in S phase but modified by phosphorylation during G2/mitosis transition. The epitope for the MPM-2 is a phosphorylated site. In this study, we attempted to determine which amino acids are phosphorylated during the G2-mitosis (M) transition. We raised a polyclonal antibody against one of the antigens recognized by MPM-2, i.e. a protein of 55 kDa, that is present in interphase cells but modified by phosphorylation during mitosis. This antibody recognizes the p55 protein in both interphase and mitosis while it is recognized by the monoclonal antibody MPM-2 only in mitotic cells. Phosphoamino acid analysis of protein p55 from 32P-labeled S-phase and M-phase HeLa cell extracts after immunoprecipitation with anti-p55 antibodies revealed that threonine was extensively phosphorylated in p55 during G2-M but not in S phase, whereas serine was phosphorylated during both S and M phases. Tyrosine was not phosphorylated. Identical results were obtained when antigens recognized by MPM-2 were subjected to similar analysis. As cells completed mitosis and entered G1 phase phosphothreonine was completely dephosphorylated whereas phosphoserine was not. These results suggest that phosphorylation of threonine might be specific to some of the mitosis-related events.     

The lamin B receptor of the inner nuclear membrane undergoes mitosis-specific phosphorylation and is a substrate for p34cdc2-type protein kinase.

The lamin B receptor (LBR) is an integral protein of the inner nuclear membrane that interacts with lamin B in vitro. If contains a 204-amino acid nucleoplasmic amino-terminal domain and a hydrophobic carboxyl-terminal domain with eight putative transmembrane segments. We found cell cycle-dependent phosphorylation of LBR using phosphoamino acid analysis and phosphopeptide mapping of in vivo 32P-labeled LBR immunoprecipitated from chicken cells in interphase and arrested in mitosis. LBR was phosphorylated only on serine residues in interphase and on serine and threonine residues in mitosis. Some serine residues phosphorylated in interphase were not phosphorylated in mitosis. To identify a threonine residue specifically phosphorylated in mitosis and the responsible protein kinase, wild-type and mutant LBR nucleoplasmic domain fusion proteins were phosphorylated in vitro by p34cdc2-type protein kinase. Comparisons of phosphopeptide maps to those of in vivo 32P-labeled mitotic LBR showed that Thr188 is likely to be phosphorylated by this enzyme during mitosis. These phosphorylation/dephosphorylation events may be responsible for some of the changes in the interaction between the nuclear lamina and the inner nuclear membrane that occur during mitosis.     

In vivo phosphorylation of the lamin B receptor. Binding of lamin B to its nuclear membrane receptor is affected by phosphorylation

Previous studies have shown that the nuclear envelope of avian erythrocytes contains a 58-kDa integral membrane protein (p58) which serves as a receptor for the karyoskeletal protein lamin B (Worman, J. H., Yuan, J., Blobel, G., and Georgatos, S. D. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 8531-8534). We now demonstrate that p58 is phosphorylated in vivo at serine residues and that its phosphorylation is stimulated by isoproterenol in a dose-dependent fashion. We further show that dephosphorylation of p58 reduces significantly its binding to lamin B. These data suggest that phosphorylation may constitute one of the major mechanisms regulating the lamina-nuclear membrane interactions.     

Phosphorylation-induced rearrangement of the histone H3 NH2-terminal domain during mitotic chromosome condensation

The NH2-terminal domain (N-tail) of histone H3 has been implicated in chromatin compaction and its phosphorylation at Ser10 is tightly correlated with mitotic chromosome condensation. We have developed one mAb that specifically recognizes histone H3 N-tails phosphorylated at Ser10 (H3P Ab) and another that recognizes phosphorylated and unphosphorylated H3 N-tails equally well (H3 Ab). Immunocytochemistry with the H3P Ab shows that Ser10 phosphorylation begins in early prophase, peaks before metaphase, and decreases during anaphase and telophase. Unexpectedly, the H3 Ab shows stronger immunofluorescence in mitosis than interphase, indicating that the H3 N-tail is more accessible in condensed mitotic chromatin than in decondensed interphase chromatin. In vivo ultraviolet laser cross-linking indicates that the H3 N-tail is bound to DNA in interphase cells and that binding is reduced in mitotic cells. Treatment of mitotic cells with the protein kinase inhibitor staurosporine causes histone H3 dephosphorylation and chromosome decondensation. It also decreases the accessibility of the H3 N-tail to H3 Ab and increases the binding of the N-tail to DNA. These results indicate that a phosphorylation-dependent weakening of the association between the H3 N-tail and DNA plays a role in mitotic chromosome condensation.     

The Dephosphorylated Form of the Anaphase-Promoting Complex Protein Cdc27/Apc3 Concentrates on Kinetochores and Chromosome Arms in Mitosis

Cell cycle regulated protein ubiquitination and degradation within subcellular domains may be essential for the normal progression of mitosis. Cdc27 is a conserved component of an essential M-phase ubiquitin-protein ligase called the anaphase-promoting complex/cyclosome. We examined the subcellular distribution of Cdc27 in greater detail in mammalian cells and found Cdc27 concentrated at spindle poles and on spindle microtubules as previously described, but also found Cdc27 at kinetochores and along chromosome arms. This localization was not dependent on intact microtubules. While the great majority of Cdc27 protein in M phase cells is highly phosphorylated, only the dephosphorylated form of Cdc27 was found associated with isolated chromosomes. Kinases that also associate with isolated chromosomes catalyzed the in vitro phosphorylation of the chromosome-associated Cdc27. Microinjection of anti-Cdc27 antibody into cells causes arrest at metaphase. Microinjection of cells with anti-Mad2 antibody normally induces premature anaphase onset resulting in catastrophic nondisjunction of the chromosomes. However, coinjection of anti-Cdc27 antibody with anti-Mad2 antibody resulted in metaphase arrest. The association of dephosphorylated APC/C components with mitotic chromosomes suggests mechanisms by which the spindle checkpoint may regulate APC/C activity at mitosis.

Key Words:

Centromere, Ubiquitin, Checkpoint, Cell cycle, Proteasome     

RanGAP1*SUMO1 is phosphorylated at the onset of mitosis and remains associated with RanBP2 upon NPC disassembly

The RanGTPase activating protein RanGAP1 has essential functions in both nucleocytoplasmic transport and mitosis. In interphase, a significant fraction of vertebrate SUMO1-modified RanGAP1 forms a stable complex with the nucleoporin RanBP2/Nup358 at nuclear pore complexes. RanBP2 not only acts in the RanGTPase cycle but also is a SUMO1 E3 ligase. Here, we show that RanGAP1 is phosphorylated on residues T409, S428, and S442. Phosphorylation occurs before nuclear envelope breakdown and is maintained throughout mitosis. Nocodazole arrest leads to quantitative phosphorylation. The M-phase kinase cyclin B/Cdk1 phosphorylates RanGAP1 efficiently in vitro, and T409 phosphorylation correlates with nuclear accumulation of cyclin B1 in vivo. We find that phosphorylated RanGAP1 remains associated with RanBP2/Nup358 and the SUMO E2-conjugating enzyme Ubc9 in mitosis, hence mitotic phosphorylation may have functional consequences for the RanGTPase cycle and/or for RanBP2-dependent sumoylation.     

Phosphorylation–dephosphorylation cycle of HP1α governs accurate mitotic progression

Heterochromatin protein 1α (HP1α), a bona fide factor of silent chromatin, is required for establishing as well as maintaining the higher-order chromatin structure in eukaryotes. HP1α is decorated with several post-translational modifications, and many of these are critical for its cellular functions. HP1α is heavily phosphorylated; however, its physiological relevance had remained to be completely understood. We have recently demonstrated that human HP1α is a mitotic target for NDR kinase, and the phosphorylation at the hinge region of HP1α at the G₂/M phase of the cell cycle is crucial for mitotic progression and Sgo1 loading at mitotic centromeres (Chakraborty et al., 2014). We now demonstrate that the dephosphorylation of HP1α within its hinge domain occurs during mitosis, specifically soon after prometaphase. In the absence of the hinge-specific HP1α phosphorylation, either as a consequence of depleting NDR1 or in cells expressing a non-phosphorylatable HP1α mutant, the cells arrest in prometaphase with several mitotic defects. In this study we show that NDR1-depleted cells expressing hinge-specific phosphomimetic HP1α mutant rescues the prometaphase arrest but displays defects in mitotic exit, suggesting that the dephosphorylation of HP1α is required for the completion of cytokinesis. Taken together, our results reveal that the phosphorylation–dephosphorylation cycle of HP1α orchestrates accurate progression of cells through mitosis.     

The Drosophila maternal-effect gene fs(1)Ya encodes a cell cycle-dependent nuclear envelope component required for embryonic mitosis

The maternal-effect gene fs(1)Ya is specifically required for embryonic mitosis in Drosophila. fs(1)Ya is involved in the initiation of the first embryonic mitosis and may also be necessary for subsequent embryonic mitotic divisions. fs(1)Ya encodes a 91.3 kd hydrophilic protein containing two putative MPF phosphorylation target sites and two potential nuclear localization signals. This protein is synthesized during postoogenic maturation from its maternal RNA and persists throughout embryogenesis. In early embryos, the fs(1)Ya protein is localized to the nuclear envelope from interphase to metaphase. During anaphase and telophase, it is dispersed in the nucleoplasm and cytoplasm, a behavior that is different from that of both the nuclear envelope and lamins. These results suggest that the fs(1)Ya protein is a cell cycle-dependent component of the nuclear envelope that specifically functions in embryonic mitosis.     

利用免疫荧光标记研究磷酸化组蛋白H3在乳腺癌细胞中的分布

在时间上与细胞周期相关并且在功能上又与染色质凝集偶联的一类组蛋白翻译后修饰就是组蛋白H3磷酸化。运用一个针对H3 Ser10磷酸化的特异性抗体 ,通过SDS PAGE、免疫印迹和免疫荧光标记检测了磷酸化H3在MCF 7细胞周期中的分布。共聚焦显微结果显示 :H3磷酸化在早前期细胞核膜附近以斑点状起始 ,之后扩展到整个凝集的染色质上 ,然后在早中期达到最高水平。H3去磷酸化开始于有丝分裂后期 ,很快在末期完成 ,而此时末期细胞凝集的染色质并未完全解凝集。H3磷酸化与染色质初期凝集之间存在着精确的时间和空间上的相关性。另外 ,对H3磷酸化可能的作用进行了讨论。     

The G2/M DNA damage checkpoint inhibits mitosis through Tyr15 phosphorylation of p34cdc2 in Aspergillus nidulans.

It is possible to cause G2 arrest in Aspergillus nidulans by inactivating either p34cdc2 or NIMA. We therefore investigated the negative control of these two mitosis-promoting kinases after DNA damage. DNA damage caused rapid Tyr15 phosphorylation of p34cdc2 and transient cell cycle arrest but had little effect on the activity of NIMA. Dividing cells deficient in Tyr15 phosphorylation of p34cdc2 were sensitive to both MMS and UV irradiation and entered lethal premature mitosis with damaged DNA. However, non-dividing quiescent conidiospores of the Tyr15 mutant strain were not sensitive to DNA damage. The UV and MMS sensitivity of cells unable to tyrosine phosphorylate p34cdc2 is therefore caused by defects in DNA damage checkpoint regulation over mitosis. Both the nimA5 and nimT23 temperature-sensitive mutations cause an arrest in G2 at 42 degrees C. Addition of MMS to nimT23 G2-arrested cells caused a marked delay in their entry into mitosis upon downshift to 32 degrees C and this delay was correlated with a long delay in the dephosphorylation and activation of p34cdc2. Addition of MMS to nimA5 G2-arrested cells caused inactivation of the H1 kinase activity of p34cdc2 due to an increase in its Tyr15 phosphorylation level and delayed entry into mitosis upon return to 32 degrees C. However, if Tyr15 phosphorylation of p34cdc2 was prevented then its H1 kinase activity was not inactivated upon MMS addition to nimA5 G2-arrested cells and they rapidly progressed into a lethal mitosis upon release to 32 degrees C. Thus, Tyr15 phosphorylation of p34cdc2 in G2 arrests initiation of mitosis after DNA damage in A. nidulans.