Key Spec Table
|Key Applications||Format||Host||Detection Methods|
|Antibody Type||Monoclonal Antibody|
|Safety Information according to GHS|
|Product Usage Statements|
|Material Size||1 mL|
References | 11 Available | See All References
|Reference overview||Pub Med ID|
|Clathrin-mediated endocytosis is inhibited during mitosis. |
Fielding, AB; Willox, AK; Okeke, E; Royle, SJ
Proceedings of the National Academy of Sciences of the United States of America 109 6572-7 2012
A long-standing paradigm in cell biology is the shutdown of endocytosis during mitosis. There is consensus that transferrin uptake is inhibited after entry into prophase and that it resumes in telophase. A recent study proposed that endocytosis is continuous throughout the cell cycle and that the observed inhibition of transferrin uptake is due to a decrease in available transferrin receptor at the cell surface, and not to a shutdown of endocytosis. This challenge to the established view is gradually becoming accepted. Because of this controversy, we revisited the question of endocytic activity during mitosis. Using an antibody uptake assay and controlling for potential changes in surface receptor density, we demonstrate the strong inhibition of endocytosis in mitosis of CD8 chimeras containing any of the three major internalization motifs for clathrin-mediated endocytosis (YXXΦ, [DE]XXXL[LI], or FXNPXY) or a CD8 protein with the cytoplasmic tail of the cation-independent mannose 6-phosphate receptor. The shutdown is not gradual: We describe a binary switch from endocytosis being "on" in interphase to "off" in mitosis as cells traverse the G(2)/M checkpoint. In addition, we show that the inhibition of transferrin uptake in mitosis occurs despite abundant transferrin receptor at the surface of HeLa cells. Our study finds no support for the recent idea that endocytosis continues during mitosis, and we conclude that endocytosis is temporarily shutdown during early mitosis.
|Rapid induction of apoptosis during Kinesin-5 inhibitor-induced mitotic arrest in HL60 cells. |
Yangzhong Tang,James D Orth,Tiao Xie,Timothy J Mitchison
Cancer letters 310 2011
Small molecule inhibitors of Kinesin-5 (K5Is) that arrest cells in mitosis with monopolar spindles are promising anti-cancer drug candidates. Clinical trials of K5Is revealed dose-limiting neutropenia, or loss of neutrophils, for which the molecular mechanism is unclear. We investigated the effects of a K5I on HL60 cells, a human promyelocytic leukemia cell line that is often used to model dividing neutrophil progenitors in cell culture. We found K5I treatment caused unusually rapid death of HL60 cells exclusively during mitotic arrest. This mitotic death occurred via the intrinsic apoptosis pathway with molecular events that include cytochrome c leakage into the cytoplasm, caspase activation, and Parp1 cleavage. Bcl-2 overexpression protected from death. We probed mitochondrial physiology to find candidate triggers of cytochrome c release, and observed a decrease of membrane potential (??m) before mitochondrial outer membrane permeabilization (MOMP). Interestingly, this loss of ??m was not blocked by overexpressing Bcl-2, suggesting it might be a cause of Bax/Bak activation, not a consequence. Taken together, these results show that K5I induces intrinsic apoptosis during mitotic arrest in HL60 with loss of ??m as an upstream event of MOMP.
|Dissecting the M phase-specific phosphorylation of serine-proline or threonine-proline motifs. |
Chuan Fen Wu,Ruoning Wang,Qianjin Liang,Jianjiao Liang,Wenke Li,Sung Yun Jung,Jun Qin,Sue-Hwa Lin,Jian Kuang
Molecular biology of the cell 21 2010
M phase induction in eukaryotic cell cycles is associated with a burst of protein phosphorylation, primarily at serine or threonine followed by proline (S/TP motif). The mitotic phosphoprotein antibody MPM-2 recognizes a significant subset of mitotically phosphorylated S/TP motifs; however, the required surrounding sequences of and the key kinases that phosphorylate these S/TP motifs remain to be determined. By mapping the mitotic MPM-2 epitopes in Xenopus Cdc25C and characterizing the mitotic MPM-2 epitope kinases in Xenopus oocytes and egg extracts, we have determined that phosphorylation of TP motifs that are surrounded by hydrophobic residues at both -1 and +1 positions plays a dominant role in M phase-associated burst of MPM-2 reactivity. Although mitotic Cdk and MAPK may phosphorylate subsets of these motifs that have a basic residue at the +2 position and a proline residue at the -2 position, respectively, the majority of these motifs that are preferentially phosphorylated in mitosis do not have these features. The M phase-associated burst of MPM-2 reactivity can be induced in Xenopus oocytes and egg extracts in the absence of MAPK or Cdc2 activity. These findings indicate that the M phase-associated burst of MPM-2 reactivity represents a novel type of protein phosphorylation in mitotic regulation.Full Text Article
|Plk1 regulates both ASAP localization and its role in spindle pole integrity. |
Grégory Eot-Houllier,Magali Venoux,Sophie Vidal-Eychenié,Minh-Thâo Hoang,Dominique Giorgi,Sylvie Rouquier
The Journal of biological chemistry 285 2010
Bipolar spindle formation is essential for faithful chromosome segregation at mitosis. Because centrosomes define spindle poles, abnormal number and structural organization of centrosomes can lead to loss of spindle bipolarity and genetic integrity. ASAP (aster-associated protein or MAP9) is a centrosome- and spindle-associated protein, the deregulation of which induces severe mitotic defects. Its phosphorylation by Aurora A is required for spindle assembly and mitosis progression. Here, we show that ASAP is localized to the spindle poles by Polo-like kinase 1 (Plk1) (a mitotic kinase that plays an essential role in centrosome regulation and mitotic spindle assembly) through the ?-TuRC-dependent pathway. We also demonstrate that ASAP is a novel substrate of Plk1 phosphorylation and have identified serine 289 as the major phosphorylation site by Plk1 in vivo. ASAP phosphorylated on serine 289 is localized to centrosomes during mitosis, but this phosphorylation is not required for its Plk1-dependent localization at the spindle poles. We show that phosphorylated ASAP on serine 289 contributes to spindle pole stability in a microtubule-dependent manner. These data reveal a novel function of ASAP in centrosome integrity. Our results highlight dual ASAP regulation by Plk1 and further confirm the importance of ASAP for spindle pole organization, bipolar spindle assembly, and mitosis.Full Text Article
|Multisite phosphorylation of Erk5 in mitosis. |
Elena Díaz-Rodríguez,Atanasio Pandiella
Journal of cell science 123 2010
The MAP kinase Erk5 plays important roles in cellular proliferation, and has recently been implicated in the regulation of mitosis. The classic pathway of Erk5 activation involves dual phosphorylation at its TEY microdomain by the upstream regulating kinase MEK5. Here we describe a second pathway that controls Erk5 phosphorylation. This pathway is activated in mitotic cells and involves kinase activities distinct from MEK5. Studies aimed at identifying these kinases suggested that CDK1 activity is required to sustain Erk5 phosphorylation in mitosis, as treatment with RO3306, a CDK1 inhibitor, reversed mitotic phosphorylation of Erk5. Moreover, CDK1 co-precipitated with Erk5 in mitotic cells. The mitotic phosphorylation of Erk5 occurs at multiple sites located at its unique C-terminal region, within an Erk5 subdomain that has formerly been implicated in the control of the subcellular location of Erk5. Furthermore, molecular studies indicated that phosphorylation at these sites may participate in the control of the transit of Erk5 between the cytosol and the nucleus, in addition to regulating its transcriptional activity. Together, our results demonstrate the existence of a second Erk5 phosphorylation pathway, that is activated in mitosis, and that may participate in the regulation of Erk5 functions.
|Calcineurin activity is required for the completion of cytokinesis. |
Megan Chircop,Chandra S Malladi,Audrey T Lian,Scott L Page,Michael Zavortink,Christopher P Gordon,Adam McCluskey,Phillip J Robinson
Cellular and molecular life sciences : CMLS 67 2010
Successful completion of cytokinesis requires the spatio-temporal regulation of protein phosphorylation and the coordinated activity of protein kinases and phosphatases. Many mitotic protein kinases are well characterized while mitotic phosphatases are largely unknown. Here, we show that the Ca(2+)- and calmodulin-dependent phosphatase, calcineurin (CaN), is required for cytokinesis in mammalian cells, functioning specifically at the abscission stage. CaN inhibitors induce multinucleation in HeLa cells and prolong the time cells spend connected via an extended intracellular bridge. Upon Ca(2+) influx during cytokinesis, CaN is activated, targeting a set of proteins for dephosphorylation, including dynamin II (dynII). At the intracellular bridge, phospho-dynII and CaN are co-localized to dual flanking midbody rings (FMRs) that reside on either side of the central midbody ring. CaN activity and disassembly of the FMRs coincide with abscission. Thus, CaN activity at the midbody plays a key role in regulating the completion of cytokinesis in mammalian cells.
|DNA damage induces Chk1-dependent threonine-160 phosphorylation and activation of Cdk2. |
E Bourke,J A L Brown,S Takeda,H Hochegger,C G Morrison
Oncogene 29 2010
Abnormal centrosome numbers arise in tumours and can cause multipolar mitoses and genome instability. Cdk2 controls normal centrosome duplication, but Chk1-dependent centrosome amplification also occurs after DNA damage. We investigated the involvement of cyclin-dependent kinases (Cdks) in DNA damage-induced centrosome amplification using cells lacking either Cdk2, or both Cdk1 and Cdk2 activity. Cdk2(-/-) DT40 cells showed robust centrosome amplification after ionizing radiation (IR), whereas Cdk1-deficient Cdk2(-/-) cells showed no centrosome amplification, demonstrating that Cdk1 can substitute for Cdk2 in this pathway. Surprisingly, we found that Cdk2 activity was upregulated by IR in wild-type but not in Chk1(-/-) DT40 cells. Cdk2 upregulation also occurred in HeLa cells after IR treatment. Chk1-dependent Cdk2 induction was not accompanied by increased levels of Cdk1, Cdk2, cyclin A or cyclin E, but activating T160 phosphorylation of Cdk2 increased after IR. Moreover, Cdk2 overexpression restored IR-induced centrosome amplification in Cdk1-deficient Cdk2(-/-) cells, but T160A mutation blocked this rescue. Our data suggest that Chk1 signalling causes centrosome amplification after IR by upregulating Cdk2 activity through activating phosphorylation.
|Differential roles of STIM1, STIM2 and Orai1 in the control of cell proliferation and SOCE amplitude in HEK293 cells. |
Charbel El Boustany,Maria Katsogiannou,Philippe Delcourt,Etienne Dewailly,Natalia Prevarskaya,Anne-Sophie Borowiec,Thierry Capiod
Cell calcium 47 2010
Orai1, together with STIM1 and STIM2, constitutes the molecular basis for store-operated calcium entry (SOCE) and we have investigated their role in cell proliferation and cell cycle progression in HEK293 cells. 48-h serum deprival, and a 24-h treatment with 1 mM hydroxyurea or with 10 microM RO-3306--a cyclin-dependent kinase 1 inhibitor--induced cell cycle block in G1, S and G2/M, respectively. SOCE amplitude, monitored in whole-cell voltage clamped cells, was markedly reduced (60-70%) in all conditions, with full reversibility within 4h. Silencing of Orai and STIM1 using siRNA resulted in a large inhibition of SOCE (70-80%) whereas siSTIM2 had a smaller but significant effect (30%). However, the cell population doubling time was not affected in siSTIM1 cells (18 h, the same as in control cells) but was increased in both siOrai1 cells (29 h) and in siSTIM2 (23 h) even when combined with siSTIM1. This suggests that STIM1 plays no role in cell proliferation in HEK293 cells while STIM2 is involved in both SOCE and cell proliferation in these cells. Finally, the cell cycle block induced SOCE inhibition was associated with reduced Orai1 expression with full recovery within 4h, whereas the expression of STIM1 and STIM2 remained unaltered. These observations reveal a tight relation between cell proliferation, calcium entry and Orai1 expression in HEK293 cells.
|APC16 is a conserved subunit of the anaphase-promoting complex/cyclosome. |
Geert J P L Kops,Monique van der Voet,Moniek van der Voet,Michael S Manak,Maria H J van Osch,Said M Naini,Andrea Brear,Ian X McLeod,Dirk M Hentschel,John R Yates,Sander van den Heuvel,Jagesh V Shah
Journal of cell science 123 2010
Error-free chromosome segregation depends on timely activation of the multi-subunit E3 ubiquitin ligase APC/C. Activation of the APC/C initiates chromosome segregation and mitotic exit by targeting critical cell-cycle regulators for destruction. The APC/C is the principle target of the mitotic checkpoint, which prevents segregation while chromosomes are unattached to spindle microtubules. We now report the identification and characterization of APC16, a conserved subunit of the APC/C. APC16 was found in association with tandem-affinity-purified mitotic checkpoint complex protein complexes. APC16 is a bona fide subunit of human APC/C: it is present in APC/C complexes throughout the cell cycle, the phenotype of APC16-depleted cells copies depletion of other APC/C subunits, and APC16 is important for APC/C activity towards mitotic substrates. APC16 sequence homologues can be identified in metazoans, but not fungi, by four conserved primary sequence stretches. We provide evidence that the C. elegans gene K10D2.4 and the D. rerio gene zgc:110659 are functional equivalents of human APC16. Our findings show that APC/C is composed of previously undescribed subunits, and raise the question of why metazoan APC/C is molecularly different from unicellular APC/C.Full Text Article
|Aurora-C kinase supports mitotic progression in the absence of Aurora-B. |
Scott D Slattery,Michael A Mancini,Bill R Brinkley,Rebecca M Hall
Cell cycle (Georgetown, Tex.) 8 2009
Aurora family kinases regulate numerous mitotic processes, and their dysfunction or overexpression can cause aneuploidy, a contributing factor for tumorigenesis. In vertebrates, the Aurora-B kinase regulates kinetochore maturation, destabilization of improper kinetochore-microtubule attachments, the spindle assembly checkpoint, central spindle organization and cytokinesis. A gene duplication event created the related Aurora-C kinase in mammals. While Aurora-C function is unclear, it has similar structural and localization properties as Aurora-B. Inhibition of either Aurora-B or Aurora-C function causes aneuploidy, while simultaneous inhibition of both causes a higher frequency of aneuploidy. To determine if Aurora-C and -B have overlapping or unique complementary functions during mitosis, we created a system where Aurora-B is replaced by wild-type or kinase-defective mutant Aurora-C in HeLa cells. In this model, Aurora-B protein levels and mitotic functions were suppressed including the regulation of kinetochore-microtubule attachments, the spindle assembly checkpoint, and cytokinesis. Wild-type, but not kinase-defective Aurora-C expression, was able to rescue these functions. Therefore, Aurora-C can perform the same essential functions as Aurora-B in mitosis.
|Distinct mechanisms act in concert to mediate cell cycle arrest. |
Jared E Toettcher,Alexander Loewer,Gerard J Ostheimer,Michael B Yaffe,Bruce Tidor,Galit Lahav
Proceedings of the National Academy of Sciences of the United States of America 106 2009
In response to DNA damage, cells arrest at specific stages in the cell cycle. This arrest must fulfill at least 3 requirements: it must be activated promptly; it must be sustained as long as damage is present to prevent loss of genomic information; and after the arrest, cells must re-enter into the appropriate cell cycle phase to ensure proper ploidy. Multiple molecular mechanisms capable of arresting the cell cycle have been identified in mammalian cells; however, it is unknown whether each mechanism meets all 3 requirements or whether they act together to confer specific functions to the arrest. To address this question, we integrated mathematical models describing the cell cycle and the DNA damage signaling networks and tested the contributions of each mechanism to cell cycle arrest and re-entry. Predictions from this model were then tested with quantitative experiments to identify the combined action of arrest mechanisms in irradiated cells. We find that different arrest mechanisms serve indispensable roles in the proper cellular response to DNA damage over time: p53-independent cyclin inactivation confers immediate arrest, whereas p53-dependent cyclin downregulation allows this arrest to be sustained. Additionally, p21-mediated inhibition of cyclin-dependent kinase activity is indispensable for preventing improper cell cycle re-entry and endoreduplication. This work shows that in a complex signaling network, seemingly redundant mechanisms, acting in a concerted fashion, can achieve a specific cellular outcome.Full Text Article
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