Barbiero, M.
Cirillo, L.
Veerapathiran, S.
Coates, C.
Ruffilli, C.
Pines, J.
(2022). Cell cycle-dependent binding between Cyclin B1 and Cdk1 revealed by time-resolved fluorescence correlation spectroscopy. ,
Vol.12
(6),
p. 220057.
show abstract
Measuring the dynamics with which the regulatory complexes assemble and disassemble is a crucial barrier to our understanding of how the cell cycle is controlled that until now has been difficult to address. This considerable gap in our understanding is due to the difficulty of reconciling biochemical assays with single cell-based techniques, but recent advances in microscopy and gene editing techniques now enable the measurement of the kinetics of protein-protein interaction in living cells. Here, we apply fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy to study the dynamics of the cell cycle machinery, beginning with Cyclin B1 and its binding to its partner kinase Cdk1 that together form the major mitotic kinase. Although Cyclin B1 and Cdk1 are known to bind with high affinity, our results reveal that in living cells there is a pool of Cyclin B1 that is not bound to Cdk1. Furthermore, we provide evidence that the affinity of Cyclin B1 for Cdk1 increases during the cell cycle, indicating that the assembly of the complex is a regulated step. Our work lays the groundwork for studying the kinetics of protein complex assembly and disassembly during the cell cycle in living cells..
Jackman, M.
Marcozzi, C.
Barbiero, M.
Pardo, M.
Yu, L.
Tyson, A.L.
Choudhary, J.S.
Pines, J.
(2020). Cyclin B1-Cdk1 facilitates MAD1 release from the nuclear pore to ensure a robust spindle checkpoint. The journal of cell biology,
Vol.219
(6).
show abstract
How the cell rapidly and completely reorganizes its architecture when it divides is a problem that has fascinated researchers for almost 150 yr. We now know that the core regulatory machinery is highly conserved in eukaryotes, but how these multiple protein kinases, protein phosphatases, and ubiquitin ligases are coordinated in space and time to remodel the cell in a matter of minutes remains a major question. Cyclin B1-Cdk is the primary kinase that drives mitotic remodeling; here we show that it is targeted to the nuclear pore complex (NPC) by binding an acidic face of the kinetochore checkpoint protein, MAD1, where it coordinates NPC disassembly with kinetochore assembly. Localized cyclin B1-Cdk1 is needed for the proper release of MAD1 from the embrace of TPR at the nuclear pore so that it can be recruited to kinetochores before nuclear envelope breakdown to maintain genomic stability..
Strauss, B.
Harrison, A.
Coelho, P.A.
Yata, K.
Zernicka-Goetz, M.
Pines, J.
(2018). Cyclin B1 is essential for mitosis in mouse embryos, and its nuclear export sets the time for mitosis. The journal of cell biology,
Vol.217
(1),
pp. 179-193.
show abstract
There is remarkable redundancy between the Cyclin-Cdk complexes that comprise the cell cycle machinery. None of the mammalian A-, D-, or E-type cyclins are required in development until implantation, and only Cdk1 is essential for early cell divisions. Cyclin B1 is essential for development, but whether it is required for cell division is contentious. Here, we used a novel imaging approach to analyze Cyclin B1-null embryos from fertilization onward. We show that Cyclin B1 -/- embryos arrest in G2 phase after just two divisions. This is the earliest arrest of any Cyclin known and places Cyclin B1 with cdk1 as the essential regulators of the cell cycle. We reintroduced mutant proteins into this genetically null background to determine why Cyclin B1 is constantly exported from the nucleus. We found that Cyclin B1 must be exported from the nucleus for the cell to prevent premature entry to mitosis, and retaining Cyclin B1-Cdk1 at the plasma membrane precludes entry to mitosis..
Yost, S.
de Wolf, B.
Hanks, S.
Zachariou, A.
Marcozzi, C.
Clarke, M.
de Voer, R.
Etemad, B.
Uijttewaal, E.
Ramsay, E.
Wylie, H.
Elliott, A.
Picton, S.
Smith, A.
Smithson, S.
Seal, S.
Ruark, E.
Houge, G.
Pines, J.
Kops, G.J.
Rahman, N.
(2017). Biallelic TRIP13 mutations predispose to Wilms tumor and chromosome missegregation. Nature genetics,
Vol.49
(7),
pp. 1148-1151.
show abstract
Through exome sequencing, we identified six individuals with biallelic loss-of-function mutations in TRIP13. All six developed Wilms tumor. Constitutional mosaic aneuploidies, microcephaly, developmental delay and seizures, which are features of mosaic variegated aneuploidy (MVA) syndrome, were more variably present. Through functional studies, we show that TRIP13-mutant patient cells have no detectable TRIP13 and have substantial impairment of the spindle assembly checkpoint (SAC), leading to a high rate of chromosome missegregation. Accurate segregation, as well as SAC proficiency, is rescued by restoring TRIP13 function. Individuals with biallelic TRIP13 or BUB1B mutations have a high risk of embryonal tumors, and here we show that their cells display severe SAC impairment. MVA due to biallelic CEP57 mutations, or of unknown cause, is not associated with embryonal tumors and cells from these individuals show minimal SAC deficiency. These data provide insights into the complex relationships between aneuploidy and carcinogenesis..
Ajduk, A.
Strauss, B.
Pines, J.
Zernicka-Goetz, M.
(2017). Delayed APC/C activation extends the first mitosis of mouse embryos. Scientific reports,
Vol.7
(1),
pp. 9682-?.
show abstract
The correct temporal regulation of mitosis underpins genomic stability because it ensures the alignment of chromosomes on the mitotic spindle that is required for their proper segregation to the two daughter cells. Crucially, sister chromatid separation must be delayed until all the chromosomes have attached to the spindle; this is achieved by the Spindle Assembly Checkpoint (SAC) that inhibits the Anaphase Promoting Complex/Cyclosome (APC/C) ubiquitin ligase. In many species the first embryonic M-phase is significantly prolonged compared to the subsequent divisions, but the reason behind this has remained unclear. Here, we show that the first M-phase in the mouse embryo is significantly extended due to a delay in APC/C activation. Unlike in somatic cells, where the APC/C first targets cyclin A2 for degradation at nuclear envelope breakdown (NEBD), we find that in zygotes cyclin A2 remains stable for a significant period of time after NEBD. Our findings that the SAC prevents cyclin A2 degradation, whereas over-expressed Plk1 stimulates it, support our conclusion that the delay in cyclin A2 degradation is caused by low APC/C activity. As a consequence of delayed APC/C activation cyclin B1 stability in the first mitosis is also prolonged, leading to the unusual length of the first M-phase..
Di Fiore, B.
Wurzenberger, C.
Davey, N.E.
Pines, J.
(2016). The Mitotic Checkpoint Complex Requires an Evolutionary Conserved Cassette to Bind and Inhibit Active APC/C. Molecular cell,
Vol.64
(6),
pp. 1144-1153.
show abstract
The Spindle Assembly Checkpoint (SAC) ensures genomic stability by preventing sister chromatid separation until all chromosomes are attached to the spindle. It catalyzes the production of the Mitotic Checkpoint Complex (MCC), which inhibits Cdc20 to inactivate the Anaphase Promoting Complex/Cyclosome (APC/C). Here we show that two Cdc20-binding motifs in BubR1 of the recently identified ABBA motif class are crucial for the MCC to recognize active APC/C-Cdc20. Mutating these motifs eliminates MCC binding to the APC/C, thereby abolishing the SAC and preventing cells from arresting in response to microtubule poisons. These ABBA motifs flank a KEN box to form a cassette that is highly conserved through evolution, both in the arrangement and spacing of the ABBA-KEN-ABBA motifs, and association with the amino-terminal KEN box required to form the MCC. We propose that the ABBA-KEN-ABBA cassette holds the MCC onto the APC/C by binding the two Cdc20 molecules in the MCC-APC/C complex..
Grallert, A.
Boke, E.
Hagting, A.
Hodgson, B.
Connolly, Y.
Griffiths, J.R.
Smith, D.L.
Pines, J.
Hagan, I.M.
(2015). A PP1–PP2A phosphatase relay controls mitotic progression. Nature,
Vol.517
(7532),
pp. 94-98.
Di Fiore, B.
Davey, N.E.
Hagting, A.
Izawa, D.
Mansfeld, J.
Gibson, T.J.
Pines, J.
(2015). The ABBA Motif Binds APC/C Activators and Is Shared by APC/C Substrates and Regulators. Developmental cell,
Vol.32
(3),
pp. 358-372.
Wieser, S.
Pines, J.
(2015). The Biochemistry of Mitosis. Cold spring harbor perspectives in biology,
Vol.7
(3),
pp. a015776-a015776.
Izawa, D.
Pines, J.
(2015). The mitotic checkpoint complex binds a second CDC20 to inhibit active APC/C. Nature,
Vol.517
(7536),
pp. 631-634.
Matsusaka, T.
Enquist-Newman, M.
Morgan, D.O.
Pines, J.
(2014). Co-activator independent differences in how the metaphase and anaphase APC/C recognise the same substrate. Biology open,
Vol.3
(10),
pp. 904-912.
show abstract
ABSTRACT
The Anaphase Promoting Complex or Cyclosome (APC/C) is critical to the control of mitosis. The APC/C is an ubiquitin ligase that targets specific mitotic regulators for proteolysis at distinct times in mitosis, but how this is achieved is not well understood. We have addressed this question by determining whether the same substrate, cyclin B1, is recognised in the same way by the APC/C at different times in mitosis. Unexpectedly, we find that distinct but overlapping motifs in cyclin B1 are recognised by the APC/C in metaphase compared with anaphase, and this does not depend on the exchange of Cdc20 for Cdh1. Thus, changes in APC/C substrate specificity in mitosis can potentially be conferred by altering interaction sites in addition to exchanging Cdc20 for Cdh1..
Collin, P.
Nashchekina, O.
Walker, R.
Pines, J.
(2013). The spindle assembly checkpoint works like a rheostat rather than a toggle switch. Nature cell biology,
Vol.15
(11),
pp. 1378-1385.
Izawa, D.
Pines, J.
(2012). Mad2 and the APC/C compete for the same site on Cdc20 to ensure proper chromosome segregation. Journal of cell biology,
Vol.199
(1),
pp. 27-37.
show abstract
The spindle assembly checkpoint (SAC) is essential to ensure proper chromosome segregation and thereby maintain genomic stability. The SAC monitors chromosome attachment, and any unattached chromosomes generate a “wait anaphase” signal that blocks chromosome segregation. The target of the SAC is Cdc20, which activates the anaphase-promoting complex/cyclosome (APC/C) that triggers anaphase and mitotic exit by ubiquitylating securin and cyclin B1. The inhibitory complex formed by the SAC has recently been shown to inhibit Cdc20 by acting as a pseudosubstrate inhibitor, but in this paper, we show that Mad2 also inhibits Cdc20 by binding directly to a site required to bind the APC/C. Mad2 and the APC/C competed for Cdc20 in vitro, and a Cdc20 mutant that does not bind stably to Mad2 abrogated the SAC in vivo. Thus, we provide insights into how Cdc20 binds the APC/C and uncover a second mechanism by which the SAC inhibits the APC/C..
Pines, J.
(2012). A red light in mitosis. Nature reviews molecular cell biology,
Vol.13
(8),
pp. 482-482.
Izawa, D.
Pines, J.
(2011). How APC/C–Cdc20 changes its substrate specificity in mitosis. Nature cell biology,
Vol.13
(3),
pp. 223-233.
Pines, J.
(2011). Cubism and the cell cycle: the many faces of the APC/C. Nature reviews molecular cell biology,
Vol.12
(7),
pp. 427-438.
Pagliuca, F.W.
Collins, M.O.
Lichawska, A.
Zegerman, P.
Choudhary, J.S.
Pines, J.
(2011). Quantitative Proteomics Reveals the Basis for the Biochemical Specificity of the Cell-Cycle Machinery. Molecular cell,
Vol.43
(3),
pp. 406-417.
Mansfeld, J.
Collin, P.
Collins, M.O.
Choudhary, J.S.
Pines, J.
(2011). APC15 drives the turnover of MCC-CDC20 to make the spindle assembly checkpoint responsive to kinetochore attachment. Nature cell biology,
Vol.13
(10),
pp. 1234-1243.
Pines, J.
Hagan, I.
(2011). The Renaissance or the cuckoo clock. Philosophical transactions of the royal society b: biological sciences,
Vol.366
(1584),
pp. 3625-3634.
show abstract
‘…in Italy, for thirty years under the Borgias, they had warfare, terror, murder and bloodshed, but they produced Michelangelo, Leonardo da Vinci and the Renaissance. In Switzerland, they had brotherly love, they had five hundred years of democracy and peace—and what did that produce? The cuckoo clock’.Orson Welles as Harry Lime:The Third ManOrson Welles might have been a little unfair on the Swiss, after all cuckoo clocks were developed in the Schwartzwald, but, more importantly, Swiss democracy gives remarkably stable government with considerable decision-making at the local level. The alternative is the battling city-states of Renaissance Italy: culturally rich but chaotic at a higher level of organization. As our understanding of the cell cycle improves, it appears that the cell is organized more along the lines of Switzerland than Renaissance Italy, and one major challenge is to determine how local decisions are made and coordinated to produce the robust cell cycle mechanisms that we observe in the cell as a whole..
Gavet, O.
Pines, J.
(2010). Progressive Activation of CyclinB1-Cdk1 Coordinates Entry to Mitosis. Developmental cell,
Vol.18
(4),
pp. 533-543.
Khodjakov, A.
Pines, J.
(2010). Centromere tension: a divisive issue. Nature cell biology,
Vol.12
(10),
pp. 919-923.
Gavet, O.
Pines, J.
(2010). Activation of cyclin B1–Cdk1 synchronizes events in the nucleus and the cytoplasm at mitosis. Journal of cell biology,
Vol.189
(2),
pp. 247-259.
show abstract
The cyclin B–Cdk1 kinase triggers mitosis in most eukaryotes. In animal cells, cyclin B shuttles between the nucleus and cytoplasm in interphase before rapidly accumulating in the nucleus at prophase, which promotes disassembly of the nuclear lamina and nuclear envelope breakdown (NEBD). What triggers the nuclear accumulation of cyclin B1 is presently unclear, although the prevailing view is that the Plk1 kinase inhibits its nuclear export. In this study, we use a biosensor specific for cyclin B1–Cdk1 activity to show that activating cyclin B1–Cdk1 immediately triggers its rapid accumulation in the nucleus through a 40-fold increase in nuclear import that remains dependent on Cdk1 activity until NEBD. Nevertheless, a substantial proportion of cyclin B1–Cdk1 remains in the cytoplasm. The increase in nuclear import is driven by changes in the nuclear import machinery that require neither Plk1 nor inhibition of nuclear export. Thus, the intrinsic link between cyclin B1–Cdk1 activation and its rapid nuclear import inherently coordinates the reorganization of the nucleus and the cytoplasm at mitotic entry..
Di Fiore, B.
Pines, J.
(2010). How cyclin A destruction escapes the spindle assembly checkpoint. Journal of cell biology,
Vol.190
(4),
pp. 501-509.
show abstract
The anaphase-promoting complex/cyclosome (APC/C) is the ubiquitin ligase essential to mitosis, which ensures that specific proteins are degraded at specific times to control the order of mitotic events. The APC/C coactivator, Cdc20, is targeted by the spindle assembly checkpoint (SAC) to restrict APC/C activity until metaphase, yet early substrates, such as cyclin A, are degraded in the presence of the active checkpoint. Cdc20 and the cyclin-dependent kinase cofactor, Cks, are required for cyclin A destruction, but how they enable checkpoint-resistant destruction has not been elucidated. In this study, we answer this problem: we show that the N terminus of cyclin A binds directly to Cdc20 and with sufficient affinity that it can outcompete the SAC proteins. Subsequently, the Cks protein is necessary and sufficient to promote cyclin A degradation in the presence of an active checkpoint by binding cyclin A–Cdc20 to the APC/C..
Pines, J.
(2009). The APC/C: A Smörgåsbord for Proteolysis. Molecular cell,
Vol.34
(2),
pp. 135-136.
Elder, A.D.
Domin, A.
Kaminski Schierle, G.S.
Lindon, C.
Pines, J.
Esposito, A.
Kaminski, C.F.
(2009). A quantitative protocol for dynamic measurements of protein interactions by Förster resonance energy transfer-sensitized fluorescence emission. Journal of the royal society interface,
Vol.6
(suppl_1).
show abstract
Fluorescence detection of acceptor molecules sensitized by Förster resonance energy transfer (FRET) is a powerful method to study protein interactions in living cells. The method requires correction for donor spectral bleed-through and acceptor cross-excitation as well as the correct normalization of signals to account for varying fluorophore concentrations and imaging parameters. In this paper, we review different methods for FRET signal normalization and then present a rigorous model for sensitized emission measurements, which is both intuitive to understand and practical to apply. The method is validated by comparison with the acceptor photobleaching and donor lifetime-imaging techniques in live cell samples containing EYFP and ECFP tandem constructs exhibiting known amounts of FRET. By varying the stoichiometry of interaction in a controlled fashion, we show that information on the fractions of interacting donors and acceptors can be recovered. Furthermore, the method is tested by performing measurements on different microscopy platforms in both widefield and confocal imaging modes to show that signals recovered under different imaging conditions are in quantitative agreement. Finally, the method is applied in the study of dynamic interactions in the cyclin–cdk family of proteins in live cells. By normalizing the obtained signals for both acceptor and donor concentrations and using a FRET exhibiting control construct for calibration, stoichiometric changes in these interactions could be visualized in real time. The paper is written to be of practical use to researchers interested in performing sensitized emission measurements. The correct interpretation of the retrieved signals in a biological context is emphasized, and guidelines are given for the practical application of the developed algorithms..
Garnett, M.J.
Mansfeld, J.
Godwin, C.
Matsusaka, T.
Wu, J.
Russell, P.
Pines, J.
Venkitaraman, A.R.
(2009). UBE2S elongates ubiquitin chains on APC/C substrates to promote mitotic exit. Nature cell biology,
Vol.11
(11),
pp. 1363-1369.
Yekezare, M.
Pines, J.
(2009). Escaping the firing squad: acetylation of BubR1 protects it from degradation in checkpoint cells. The embo journal,
Vol.28
(14),
pp. 1991-1993.
Ahel, I.
Ahel, D.
Matsusaka, T.
Clark, A.J.
Pines, J.
Boulton, S.J.
West, S.C.
(2008). Poly(ADP-ribose)-binding zinc finger motifs in DNA repair/checkpoint proteins. Nature,
Vol.451
(7174),
pp. 81-85.
Wolthuis, R.
Clay-Farrace, L.
van Zon, W.
Yekezare, M.
Koop, L.
Ogink, J.
Medema, R.
Pines, J.
(2008). Cdc20 and Cks Direct the Spindle Checkpoint-Independent Destruction of Cyclin A. Molecular cell,
Vol.30
(3),
pp. 290-302.
Di Fiore, B.
Pines, J.
(2008). Defining the role of Emi1 in the DNA replication–segregation cycle. Chromosoma,
Vol.117
(4),
pp. 333-338.
Floyd, S.
Pines, J.
Lindon, C.
(2008). APC/CCdh1 Targets Aurora Kinase to Control Reorganization of the Mitotic Spindle at Anaphase. Current biology,
Vol.18
(21),
pp. 1649-1658.
Nilsson, J.
Yekezare, M.
Minshull, J.
Pines, J.
(2008). The APC/C maintains the spindle assembly checkpoint by targeting Cdc20 for destruction. Nature cell biology,
Vol.10
(12),
pp. 1411-1420.
Walker, A.
Acquaviva, C.
Matsusaka, T.
Koop, L.
Pines, J.
(2008). UbcH10 has a rate-limiting role in G1 phase but might not act in the spindle checkpoint or as part of an autonomous oscillator. Journal of cell science,
Vol.121
(14),
pp. 2319-2326.
show abstract
Ubiquitin-dependent proteolysis mediated by the anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase lies at the heart of the cell cycle. The APC/C targets mitotic cyclins for destruction in mitosis and G1 phase and is then inactivated at S phase, thereby generating the alternating states of high and low cyclin-Cdk activity required for the alternation of mitosis and DNA replication. Two key questions are how the APC/C is held in check by the spindle-assembly checkpoint to delay cells in mitosis in the presence of improperly attached chromosomes, and how the APC/C is inactivated once cells exit mitosis. The ubiquitin-conjugating protein UbcH10 has been proposed to be crucial in the answers to both questions. However, here we show that the behaviour of UbcH10 is inconsistent with both a crucial role in the spindle checkpoint and in inactivating the APC/C as part of an autonomous oscillator. Instead, we find that the rate-limiting role of UbcH10 is only at the end of G1 phase, just before DNA replication begins..
Basto, R.
Pines, J.
(2007). The Centrosome Opens the Way to Mitosis. Developmental cell,
Vol.12
(4),
pp. 475-477.
Di Fiore, B.
Pines, J.
(2007). Emi1 is needed to couple DNA replication with mitosis but does not regulate activation of the mitotic APC/C. Journal of cell biology,
Vol.177
(3),
pp. 425-437.
show abstract
Ubiquitin-mediated proteolysis is critical for the alternation between DNA replication and mitosis and for the key regulatory events in mitosis. The anaphase-promoting complex/cyclosome (APC/C) is a conserved ubiquitin ligase that has a fundamental role in regulating mitosis and the cell cycle in all eukaryotes. In vertebrate cells, early mitotic inhibitor 1 (Emi1) has been proposed as an important APC/C inhibitor whose destruction may trigger activation of the APC/C at mitosis. However, in this study, we show that the degradation of Emi1 is not required to activate the APC/C in mitosis. Instead, we uncover a key role for Emi1 in inhibiting the APC/C in interphase to stabilize the mitotic cyclins and geminin to promote mitosis and prevent rereplication. Thus, Emi1 plays a crucial role in the cell cycle to couple DNA replication with mitosis, and our results also question the current view that the APC/C has to be inactivated to allow DNA replication..
Pines, J.
(2006). New approaches to research on mitosis. Methods,
Vol.38
(1),
pp. 1-1.
Pines, J.
(2006). Mitosis: a matter of getting rid of the right protein at the right time. Trends in cell biology,
Vol.16
(1),
pp. 55-63.
Dasso, M.
Ohno, M.
Pines, J.
Stewart, M.
(2006). Meeting Report: International Symposium on Ran and the Cell Cycle, October 2-5, 2005, Awaji Yumebutai, Japan. Traffic,
Vol.7
(4),
pp. 474-478.
Acquaviva, C.
Pines, J.
(2006). The anaphase-promoting complex/cyclosome: APC/C. Journal of cell science,
Vol.119
(12),
pp. 2401-2404.
Pines, J.
Lindon, C.
(2005). Proteolysis: anytime, any place, anywhere?. Nature cell biology,
Vol.7
(8),
pp. 731-735.
Acquaviva, C.
Herzog, F.
Kraft, C.
Pines, J.
(2004). The anaphase promoting complex/cyclosome is recruited to centromeres by the spindle assembly checkpoint. Nature cell biology,
Vol.6
(9),
pp. 892-898.
Matsusaka, T.
Pines, J.
(2004). Chfr acts with the p38 stress kinases to block entry to mitosis in mammalian cells. Journal of cell biology,
Vol.166
(4),
pp. 507-516.
show abstract
Entry into mitosis in vertebrate cells is guarded by a checkpoint that can be activated by a variety of insults, including chromosomal damage and disrupting microtubules (Rieder, C.L., and R.W. Cole. 1998. J. Cell Biol. 142:1013–1022; Rieder, C.L., and R.W. Cole. 2000. Curr. Biol. 10:1067–1070). This checkpoint acts at the end of interphase to delay cells from entering mitosis, causing cells in prophase to decondense their chromosomes and return to G2 phase. Here, we show that in response to microtubule poisons this “antephase” checkpoint is primarily mediated by the p38 stress kinases and requires the Chfr protein that is absent or inactive in several transformed cell lines (Scolnick, D.M., and T.D. Halazonetis. 2000. Nature. 406:430–435) and lung tumors (Mizuno, K., H. Osada, H. Konishi, Y. Tatematsu, Y. Yatabe, T. Mitsudomi, Y. Fujii, and T. Takahashi. 2002. Oncogene. 21:2328–2333). Furthermore, in contrast to previous reports, we find that the checkpoint requires ubiquitylation but not proteasome activity, which is in agreement with the recent demonstration that Chfr conjugates ubiquitin through lysine 63 and not lysine 48 (Bothos, J., M.K. Summers, M. Venere, D.M. Scolnick, and T.D. Halazonetis. 2003. Oncogene. 22:7101–7107)..
Lindon, C.
Pines, J.
(2004). Ordered proteolysis in anaphase inactivates Plk1 to contribute to proper mitotic exit in human cells. Journal of cell biology,
Vol.164
(2),
pp. 233-241.
show abstract
We have found that key mitotic regulators show distinct patterns of degradation during exit from mitosis in human cells. Using a live-cell assay for proteolysis, we show that two of these regulators, polo-like kinase 1 (Plk1) and Aurora A, are degraded at different times after the anaphase-promoting complex/cyclosome (APC/C) switches from binding Cdc20 to Cdh1. Therefore, events in addition to the switch from Cdc20 to Cdh1 control the proteolysis of APC/CCdh1 substrates in vivo. We have identified a putative destruction box in Plk1 that is required for degradation of Plk1 in anaphase, and have examined the effect of nondegradable Plk1 on mitotic exit. Our results show that Plk1 proteolysis contributes to the inactivation of Plk1 in anaphase, and that this is required for the proper control of mitotic exit and cytokinesis. Our experiments reveal a role for APC/C-mediated proteolysis in exit from mitosis in human cells..
Jackman, M.
Lindon, C.
Nigg, E.A.
Pines, J.
(2003). Active cyclin B1–Cdk1 first appears on centrosomes in prophase. Nature cell biology,
Vol.5
(2),
pp. 143-148.
Clay-Farrace, L.
(2003). Human replication protein Cdc6 prevents mitosis through a checkpoint mechanism that implicates Chk1. The embo journal,
Vol.22
(3),
pp. 704-712.
Kornbluth, S.
Pines, J.
(2003). Cell division, growth and death. Current opinion in cell biology,
Vol.15
(6),
pp. 645-647.
Kraft, C.
(2003). Mitotic regulation of the human anaphase-promoting complex by phosphorylation. The embo journal,
Vol.22
(24),
pp. 6598-6609.
Pines, J.
(2002). Cell cycle trials in Salamanca. Embo reports,
Vol.3
(1),
pp. 17-21.
Jackman, M.
Kubota, Y.
den Elzen, N.
Hagting, A.
Pines, J.
(2002). Cyclin A- and Cyclin E-Cdk Complexes Shuttle between the Nucleus and the Cytoplasm. Molecular biology of the cell,
Vol.13
(3),
pp. 1030-1045.
show abstract
Cyclins A and E and their partner cyclin-dependent kinases (Cdks) are key regulators of DNA synthesis and of mitosis. Immunofluorescence studies have shown that both cyclins are nuclear and that a proportion of cyclin A is localized to sites of DNA replication. However, recently, both cyclin A and cyclin E have been implicated as regulators of centrosome replication, and it is unclear when and where these cyclin-Cdks can interact with cytoplasmic substrates. We have used live cell imaging to study the behavior of cyclin/Cdk complexes. We found that cyclin A and cyclin E are able to regulate both nuclear and cytoplasmic events because they both shuttle between the nucleus and the cytoplasm. However, we found that there are marked differences in their shuttling behavior, which raises the possibility that cyclin/Cdk function could be regulated at the level of nuclear import and export. In the course of these experiments, we have also found that, contrary to published results, mutations in the hydrophobic patch of cyclin A do affect Cdk binding and nuclear import. This has implications for the role of the hydrophobic patch as a substrate selection motif..
Nguyen, T.B.
Manova, K.
Capodieci, P.
Lindon, C.
Bottega, S.
Wang, X.-.
Refik-Rogers, J.
Pines, J.
Wolgemuth, D.J.
Koff, A.
(2002). Characterization and Expression of Mammalian Cyclin B3, a Prepachytene Meiotic Cyclin. Journal of biological chemistry,
Vol.277
(44),
pp. 41960-41969.
Hagting, A.
den Elzen, N.
Vodermaier, H.C.
Waizenegger, I.C.
Peters, J.-.
Pines, J.
(2002). Human securin proteolysis is controlled by the spindle checkpoint and reveals when the APC/C switches from activation by Cdc20 to Cdh1. Journal of cell biology,
Vol.157
(7),
pp. 1125-1137.
show abstract
Progress through mitosis is controlled by the sequential destruction of key regulators including the mitotic cyclins and securin, an inhibitor of anaphase whose destruction is required for sister chromatid separation. Here we have used live cell imaging to determine the exact time when human securin is degraded in mitosis. We show that the timing of securin destruction is set by the spindle checkpoint; securin destruction begins at metaphase once the checkpoint is satisfied. Furthermore, reimposing the checkpoint rapidly inactivates securin destruction. Thus, securin and cyclin B1 destruction have very similar properties. Moreover, we find that both cyclin B1 and securin have to be degraded before sister chromatids can separate. A mutant form of securin that lacks its destruction box (D-box) is still degraded in mitosis, but now this is in anaphase. This destruction requires a KEN box in the NH2 terminus of securin and may indicate the time in mitosis when ubiquitination switches from APCCdc20 to APCCdh1. Lastly, a D-box mutant of securin that cannot be degraded in metaphase inhibits sister chromatid separation, generating a cut phenotype where one cell can inherit both copies of the genome. Thus, defects in securin destruction alter chromosome segregation and may be relevant to the development of aneuploidy in cancer..
Okuno, Y.
(2001). Stability, chromatin association and functional activity of mammalian pre-replication complex proteins during the cell cycle. The embo journal,
Vol.20
(15),
pp. 4263-4277.
Pines, J.
Rieder, C.L.
(2001). Re-staging mitosis: a contemporary view of mitotic progression. Nature cell biology,
Vol.3
(1),
pp. E3-E6.
den Elzen, N.
Pines, J.
(2001). Cyclin a Is Destroyed in Prometaphase and Can Delay Chromosome Alignment and Anaphase. Journal of cell biology,
Vol.153
(1),
pp. 121-136.
show abstract
Mitosis is controlled by the specific and timely degradation of key regulatory proteins, notably the mitotic cyclins that bind and activate the cyclin-dependent kinases (Cdks). In animal cells, cyclin A is always degraded before cyclin B, but the exact timing and the mechanism underlying this are not known. Here we use live cell imaging to show that cyclin A begins to be degraded just after nuclear envelope breakdown. This degradation requires the 26S proteasome, but is not affected by the spindle checkpoint. Neither deletion of its destruction box nor disrupting Cdk binding prevents cyclin A proteolysis, but Cdk binding is necessary for degradation at the correct time. We also show that increasing the levels of cyclin A delays chromosome alignment and sister chromatid segregation. This delay depends on the proteolysis of cyclin A and is not caused by a lag in the bipolar attachment of chromosomes to the mitotic spindle, nor is it mediated via the spindle checkpoint. Thus, proteolysis that is not under the control of the spindle checkpoint is required for chromosome alignment and anaphase..
Draviam, V.M.
Orrechia, S.
Lowe, M.
Pardi, R.
Pines, J.
(2001). The Localization of Human Cyclins B1 and B2 Determines Cdk1 Substrate Specificity and Neither Enzyme Requires Mek to Disassemble the Golgi Apparatus. Journal of cell biology,
Vol.152
(5),
pp. 945-958.
show abstract
In this paper, we show that substrate specificity is primarily conferred on human mitotic cyclin-dependent kinases (CDKs) by their subcellular localization. The difference in localization of the B-type cyclin–CDKs underlies the ability of cyclin B1–CDK1 to cause chromosome condensation, reorganization of the microtubules, and disassembly of the nuclear lamina and of the Golgi apparatus, while it restricts cyclin B2–CDK1 to disassembly of the Golgi apparatus. We identify the region of cyclin B2 responsible for its localization and show that this will direct cyclin B1 to the Golgi apparatus and confer upon it the more limited properties of cyclin B2. Equally, directing cyclin B2 to the cytoplasm with the NH2 terminus of cyclin B1 confers the broader properties of cyclin B1. Furthermore, we show that the disassembly of the Golgi apparatus initiated by either mitotic cyclin–CDK complex does not require mitogen-activated protein kinase kinase (MEK) activity..
Zernicka-Goetz, M.
Pines, J.
(2001). Use of Green Fluorescent Protein in Mouse Embryos. Methods,
Vol.24
(1),
pp. 55-60.
Ko, T.K.
Kelly, E.
Pines, J.
(2001). CrkRS: a novel conserved Cdc2-related protein kinase that colocalises with SC35 speckles. Journal of cell science,
Vol.114
(14),
pp. 2591-2603.
Miska, E.A.
(2001). Differential localization of HDAC4 orchestrates muscle differentiation. Nucleic acids research,
Vol.29
(16),
pp. 3439-3447.
Ko, T.K.
Kelly, E.
Pines, J.
(2001). CrkRS. Journal of cell science,
Vol.114
(14),
pp. 2591-2603.
show abstract
We have isolated and characterised a novel human protein kinase, Cdc2-related kinase with an arginine/serine-rich (RS) domain (CrkRS), that is most closely related to the cyclin-dependent kinase (CDK) family. CrkRS is a 1490 amino acid protein, the largest CDK-related kinase so far isolated. The protein kinase domain of CrkRS is 89% identical to the 46 kDa CHED protein kinase, but outside the kinase domains the two proteins are completely unrelated. CrkRS has extensive proline-rich regions that match the consensus for SH3 and WW domain binding sites, and an RS domain that is predominantly found in splicing factors. CrkRS is ubiquitously expressed in tissues, and maps to a single genetic locus. There are closely related protein kinases in both the Drosophila and Caenorhabditis elegans genomes. Consistent with the presence of an RS domain, anti-CrkRS antibodies stain nuclei in a speckled pattern, overlapping with spliceosome components and the hyperphosphorylated form of RNA polymerase II. Like RNA polymerase II, CrkRS is a constitutive MPM-2 antigen throughout the cell cycle. Anti-CrkRS immunoprecipitates phosphorylate the C-terminal domain of RNA polymerase II in vitro. Thus CrkRS may be a novel, conserved link between the transcription and splicing machinery..
Pines, J.
(1999). Checkpoint on the nuclear frontier. Nature,
Vol.397
(6715),
pp. 104-105.
Clute, P.
Pines, J.
(1999). Temporal and spatial control of cyclin B1 destruction in metaphase. Nature cell biology,
Vol.1
(2),
pp. 82-87.
Hagting, A.
Jackman, M.
Simpson, K.
Pines, J.
(1999). Translocation of cyclin B1 to the nucleus at prophase requires a phosphorylation-dependent nuclear import signal. Current biology,
Vol.9
(13),
pp. 680-689.
Pines, J.
(1999). Four-dimensional control of the cell cycle. Nature cell biology,
Vol.1
(3),
pp. E73-E79.
Karlsson, C.
Katich, S.
Hagting, A.
Hoffmann, I.
Pines, J.
(1999). Cdc25b and Cdc25c Differ Markedly in Their Properties as Initiators of Mitosis. Journal of cell biology,
Vol.146
(3),
pp. 573-584.
show abstract
We have used time-lapse fluorescence microscopy to study the properties of the Cdc25B and Cdc25C phosphatases that have both been implicated as initiators of mitosis in human cells. To differentiate between the functions of the two proteins, we have microinjected expression constructs encoding Cdc25B or Cdc25C or their GFP-chimeras into synchronized tissue culture cells. This assay allows us to express the proteins at defined points in the cell cycle. We have followed the microinjected cells by time-lapse microscopy, in the presence or absence of DNA synthesis inhibitors, and assayed whether they enter mitosis prematurely or at the correct time. We find that overexpressing Cdc25B alone rapidly causes S phase and G2 phase cells to enter mitosis, whether or not DNA replication is complete, whereas overexpressing Cdc25C does not cause premature mitosis. Overexpressing Cdc25C together with cyclin B1 does shorten the G2 phase and can override the unreplicated DNA checkpoint, but much less efficiently than overexpressing Cdc25B. These results suggest that Cdc25B and Cdc25C do not respond identically to the same cell cycle checkpoints. This difference may be related to the differential localization of the proteins; Cdc25C is nuclear throughout interphase, whereas Cdc25B is nuclear in the G1 phase and cytoplasmic in the S and G2 phases. We have found that the change in subcellular localization of Cdc25B is due to nuclear export and that this is dependent on cyclin B1. Our data suggest that although both Cdc25B and Cdc25C can promote mitosis, they are likely to have distinct roles in the controlling the initiation of mitosis..
Miska, E.A.
(1999). HDAC4 deacetylase associates with and represses the MEF2 transcription factor. The embo journal,
Vol.18
(18),
pp. 5099-5107.
Furuno, N.
den Elzen, N.
Pines, J.
(1999). Human Cyclin a Is Required for Mitosis until Mid Prophase. Journal of cell biology,
Vol.147
(2),
pp. 295-306.
show abstract
We have used microinjection and time-lapse video microscopy to study the role of cyclin A in mitosis. We have injected purified, active cyclin A/cyclin-dependent kinase 2 (CDK2) into synchronized cells at specific points in the cell cycle and assayed its effect on cell division. We find that cyclin A/CDK2 will drive G2 phase cells into mitosis within 30 min of microinjection, up to 4 h before control cells enter mitosis. Often this premature mitosis is abnormal; the chromosomes do not completely condense and daughter cells fuse. Remarkably, microinjecting cyclin A/CDK2 into S phase cells has no effect on progress through the following G2 phase or mitosis. In complementary experiments we have microinjected the amino terminus of p21Cip1/Waf1/Sdi1 (p21N) into cells to inhibit cyclin A/CDK2 activity. We find that p21N will prevent S phase or G2 phase cells from entering mitosis, and will cause early prophase cells to return to interphase. These results suggest that cyclin A/CDK2 is a rate-limiting component required for entry into mitosis, and for progress through mitosis until late prophase. They also suggest that cyclin A/CDK2 may be the target of the recently described prophase checkpoint..
Arnaud, L.
Pines, J.
Nigg, E.A.
(1998). GFP tagging reveals human Polo-like kinase 1 at the kinetochore/centromere region of mitotic chromosomes. Chromosoma,
Vol.107
(6-7),
pp. 424-429.
Pines, J.
Jackman, M.
Simpson, K.
(1998). Assays for
CDK
Activity and
DNA
Replication in the Cell Cycle. Current protocols in cell biology,
Vol.00
(1).
Hagting, A.
Karlsson, C.
Clute, P.
Jackman, M.
Pines, J.
(1998). MPF localization is controlled by nuclear export. The embo journal,
Vol.17
(14),
pp. 4127-4138.
Pines, J.
(1998). Regulation of the G2 to M Transition. ,
,
pp. 57-78.
Krude, T.
Jackman, M.
Pines, J.
Laskey, R.A.
(1997). Cyclin/Cdk-Dependent Initiation of DNA Replication in a Human Cell-Free System. Cell,
Vol.88
(1),
pp. 109-119.
Pines, J.
(1997). Cyclin-dependent kinase inhibitors: the age of crystals. Biochimica et biophysica acta (bba) - reviews on cancer,
Vol.1332
(1),
pp. M39-M42.
Zernicka-Goetz, M.
Pines, J.
Hunter McLean, S.
Dixon, J.P.
Siemering, K.R.
Haseloff, J.
Evans, M.J.
(1997). Following cell fate in the living mouse embryo. Development,
Vol.124
(6),
pp. 1133-1137.
show abstract
It has been difficult to follow many of the dramatic changes in cell fate and cell migration during mouse development. This is because there has been no enduring marker that would allow cells to be recognised in the living embryo. We believe that we have overcome this problem by developing a novel form of green fluorescent protein, named MmGFP, that proves to be easily visible and non toxic to mouse cells and does not perturb embryogenesis. We show that synthetic mRNA encoding MmGFP can be injected into blastomeres to follow the fate of their progeny during preimplantation development. We have made a stable embryonic stem cell line that expresses MmGFP and introduced these fluorescent cells into mouse embryos. For the first time, we have been able to follow the fate of embryonic stem cells in living embryos and to observe directly the contribution of these cells to distinct lineages of the postimplantation embryo. This approach should lead to a more complete description of the dynamics of cell fate in the mouse..
Jackman, M.R.
Pines, J.N.
(1997). Cyclins and the G(2)/M transition. Cancer surveys,
Vol.29,
pp. 47-73.
Pines, J.
(1997). Localization of cell cycle regulators by immunofluorescence. ,
,
pp. 99-113.
Pines, J.
(1996). Cyclin from Sea Urchins to HeLas: Making the Human Cell Cycle. Biochemical society transactions,
Vol.24
(1),
pp. 15-33.
Zernicka-Goetz, M.
Pines, J.
Ryan, K.
Siemering, K.R.
Haseloff, J.
Evans, M.J.
Gurdon, J.B.
(1996). An indelible lineage marker for Xenopus using a mutated green fluorescent protein. Development,
Vol.122
(12),
pp. 3719-3724.
show abstract
We describe the use of a DNA construct (named GFP.RN3) encoding green fluorescent protein as a lineage marker for Xenopus embryos. This offers the following advantages over other lineage markers so far used in Xenopus. When injected as synthetic mRNA, its protein emits intense fluorescence in living embryos. It is non-toxic, and the fluorescence does not bleach when viewed under 480 nm light. It is surprisingly stable, being strongly visible up to the feeding tadpole stage (5 days), and in some tissues for several weeks after mRNA injection. We also describe a construct that encodes a blue fluorescent protein. We exemplify the use of this GFP.RN3 construct for marking the lineage of individual blastomeres at the 32- to 64-cell stage, and as a marker for single transplanted blastula cells. Both procedures have revealed that the descendants of one embryonic cell can contribute single muscle cells to nearly all segmental myotomes rather than predominantly to any one myotome. An independent aim of our work has been to follow the fate of cells in which an early regulatory gene has been temporarily overexpressed. For this purpose, we co-injected GFP.RN3 mRNA and mRNA for the early Xenopus gene Eomes, and found that a high concentration of Eomes results in ectopic muscle gene activation in only the injected cells. This marker may therefore be of general value in providing long term identification of those cells in which an early gene with ephemeral expression has been overexpressed..
Pines, J.
(1996). Cell cycle: Reaching for a role for the Cks proteins. Current biology,
Vol.6
(11),
pp. 1399-1402.
Jackman, M.
Firth, M.
Pines, J.
(1995). Human cyclins B1 and B2 are localized to strikingly different structures: B1 to microtubules, B2 primarily to the Golgi apparatus. The embo journal,
Vol.14
(8),
pp. 1646-1654.
Pines, J.
(1995). Cyclins, CDKs and cancer. Seminars in cancer biology,
Vol.6
(2),
pp. 63-72.
Pines, J.
(1995). Confirmational change. Nature,
Vol.376
(6538),
pp. 294-295.
Pines, J.
(1995). GFP in mammalian cells. Trends in genetics,
Vol.11
(8),
pp. 326-327.
Pines, J.
(1995). Cyclins and cyclin-dependent kinases: a biochemical view. Biochemical journal,
Vol.308
(3),
pp. 697-711.
Pines, J.
(1995). Cyclins and Cyclin-Dependent Kinases: Theme and Variations. ,
,
pp. 181-212.
Pines, J.
(1994). Arresting developments in cell-cycle control. Trends in biochemical sciences,
Vol.19
(4),
pp. 143-145.
Pines, J.
(1994). p21 inhibits cyclin shock. Nature,
Vol.369
(6481),
pp. 520-521.
Pines, J.
Hunter, T.
(1994). The differential localization of human cyclins A and B is due to a cytoplasmic retention signal in cyclin B. The embo journal,
Vol.13
(16),
pp. 3772-3781.
PINES, J.
(1994). THE CELL-CYCLE KINASES. Seminars in cancer biology,
Vol.5
(4),
pp. 305-313.
Pines, J.
(1994). Ubiquitin with everything. Nature,
Vol.371
(6500),
pp. 742-743.
Hunter, T.
Pines, J.
(1994). Cyclins and cancer II: Cyclin D and CDK inhibitors come of age. Cell,
Vol.79
(4),
pp. 573-582.
Pines, J.
(1994). Protein kinases and cell cycle control. Seminars in cell biology,
Vol.5
(6),
pp. 399-408.
Pines, J.
(1993). Cyclins and their associated cyclin-dependent kinases in the human cell cycle. Biochemical society transactions,
Vol.21
(4),
pp. 921-925.
O'Connor, P.M.
Ferris, D.K.
Pagano, M.
Draetta, G.
Pines, J.
Hunter, T.
Longo, D.L.
Kohn, K.W.
(1993). G2 delay induced by nitrogen mustard in human cells affects cyclin A/cdk2 and cyclin B1/cdc2-kinase complexes differently. Journal of biological chemistry,
Vol.268
(11),
pp. 8298-8308.
Pines, J.
(1993). Cyclins and cyclin-dependent kinases: take your partners. Trends in biochemical sciences,
Vol.18
(6),
pp. 195-197.
Pines, J.
(1993). Clear as crystal?. Current biology,
Vol.3
(8),
pp. 544-547.
Hamaguchi, J.R.
Tobey, R.A.
Pines, J.
Crissman, H.A.
Hunter, T.
Bradbury, E.M.
(1992). Requirement for p34cdc2 kinase is restricted to mitosis in the mammalian cdc2 mutant FT210. Journal of cell biology,
Vol.117
(5),
pp. 1041-1053.
show abstract
The mouse FT210 cell line is a temperature-sensitive cdc2 mutant. FT210 cells are found to arrest specifically in G2 phase and unlike many alleles of cdc2 and cdc28 mutants of yeasts, loss of p34cdc2 at the nonpermissive temperature has no apparent effect on cell cycle progression through the G1 and S phases of the division cycle. FT210 cells and the parent wild-type FM3A cell line each possess at least three distinct histone H1 kinases. H1 kinase activities in chromatography fractions were identified using a synthetic peptide substrate containing the consensus phosphorylation site of histone H1 and the kinase subunit compositions were determined immunochemically with antisera prepared against the "PSTAIR" peptide, the COOH-terminus of mammalian p34cdc2 and the human cyclins A and B1. The results show that p34cdc2 forms two separate complexes with cyclin A and with cyclin B1, both of which exhibit thermal lability at the non-permissive temperature in vitro and in vivo. A third H1 kinase with stable activity at the nonpermissive temperature is comprised of cyclin A and a cdc2-like 34-kD subunit, which is immunoreactive with anti-"PSTAIR" antiserum but is not recognized with antiserum specific for the COOH-terminus of p34cdc2. The cyclin A-associated kinases are active during S and G2 phases and earlier in the division cycle than the p34cdc2-cyclin B1 kinase. We show that mouse cells possess at least two cdc2-related gene products which form cell cycle regulated histone H1 kinases and we propose that the murine homolog of yeast p34cdc/CDC28 is essential only during the G2-to-M transition in FT210 cells..
PINES, J.
HUNTER, T.
(1992). CYCLIN-A AND CYCLIN-B1 IN THE HUMAN CELL-CYCLE. Ciba foundation symposia,
Vol.170,
pp. 187-18.
Bailly, E.
Pines, J.
Hunter, T.
Bornens, M.
(1992). Cytoplasmic accumulation of cyclin B1 in human cells: association with a detergent-resistant compartment and with the centrosome. Journal of cell science,
Vol.101
(3),
pp. 529-545.
show abstract
Mitotic cyclins are thought to function as key regulatory subunits of the universal M-phase-promoting p34cdc2 protein kinase. Previous immunolocalization studies have demonstrated that a fraction of p34cdc2 undergoes cell cycle-dependent accumulation at the centrosome during G2/M. In order to identify the mitotic cyclins with which this p34cdc2 fraction interacts, we carefully examined the subcellular distribution of both cyclin A and B1 in HeLa cells. We show here that part of cyclin B1 is associated with duplicating centrosomes throughout its accumulation in the cytoplasm and up to metaphase. In contrast cyclin A does not exhibit centrosomal association except at the onset of mitosis, from preprophase up to metaphase. We also present cytological and biochemical evidence that cyclin B1 is preferentially accumulated as a detergent-insoluble protein independently of the state of microtubule assembly and under experimental conditions where most of p34cdc2 is soluble. Interestingly, the electrophoretic pattern of the minor insoluble p34cdc2 fraction was previously shown to be particularly enriched in slow-migrating and presumably hyperphosphorylated isoforms, known to interact specifically with cyclin B1 during interphase. From these results we propose that the interaction of cyclin B1 with the centrosomes and with the cytoplasmic structures is a constitutive feature of the mechanism whereby a fraction of p34cdc2 is recruited and subsequently targeted to the cyclin B-dependent activation pathway..
Devoto, S.H.
Mudryj, M.
Pines, J.
Hunter, T.
Nevins, J.R.
(1992). A cyclin A-protein kinase complex possesses sequence-specific DNA binding activity: p33cdk2 is a component of the E2F-cyclin A complex. Cell,
Vol.68
(1),
pp. 167-176.
OCONNOR, P.M.
FERRIS, D.K.
WHITE, G.A.
PINES, J.
HUNTER, T.
LONGO, D.L.
KOHN, K.W.
(1992). RELATIONSHIPS BETWEEN CDC2 KINASE, DNA CROSS-LINKING, AND CELL-CYCLE PERTURBATIONS INDUCED BY NITROGEN-MUSTARD. Cell growth & differentiation,
Vol.3
(1),
pp. 43-52.
Lu, X.P.
Koch, K.S.
Lew, D.J.
Dulic, V.
Pines, J.
Reed, S.I.
Hunter, T.
Leffert, H.L.
(1992). Induction of cyclin mRNA and cyclin-associated histone H1 kinase during liver regeneration. Journal of biological chemistry,
Vol.267
(5),
pp. 2841-2844.
Pines, J.
(1992). Cell proliferation and control. Current opinion in cell biology,
Vol.4
(2),
pp. 144-148.
Lock, L.F.
Pines, J.
Hunter, T.
Gilbert, D.J.
Gopalan, G.
Jenkins, N.A.
Copeland, N.G.
Donovan, P.J.
(1992). A single cyclin A gene and multiple cyclin B1-related sequences are dispersed in the mouse genome. Genomics,
Vol.13
(2),
pp. 415-424.
PINES, J.
HUNTER, T.
(1992). CYCLIN-A AND CYCLIN-B1 IN THE HUMAN CELL-CYCLE. Ciba foundation symposia,
Vol.170,
pp. 187-204.
Pines, J.
Hunter, T.
(1991). Human Cell Division: The Involvement of Cyclins A and B1, and Multiple cdc2s. Cold spring harbor symposia on quantitative biology,
Vol.56
(0),
pp. 449-463.
Pines, J.
Hunter, T.
(1991). Human cyclins A and B1 are differentially located in the cell and undergo cell cycle-dependent nuclear transport. Journal of cell biology,
Vol.115
(1),
pp. 1-17.
show abstract
We have used immunofluorescence staining to study the subcellular distribution of cyclin A and B1 during the somatic cell cycle. In both primary human fibroblasts and in epithelial tumor cells, we find that cyclin A is predominantly nuclear from S phase onwards. Cyclin A may associated with condensing chromosomes in prophase, but is not associated with condensed chromosomes in metaphase. By contrast, cyclin B1 accumulates in the cytoplasm of interphase cells and only enters the nucleus at the beginning of mitosis, before nuclear lamina breakdown. In mitotic cells, cyclin B1 associates with condensed chromosomes in prophase and metaphase, and with the mitotic apparatus. Cyclin A is degraded during metaphase and cyclin B1 is precipitously destroyed at the metaphase----anaphase transition. Cell fractionation and immunoprecipitation studies showed that both cyclin A and cyclin B1 are associated with PSTAIRE-containing proteins. The nuclear, but not the cytoplasmic form, of cyclin A is associated with a 33-kD PSTAIRE-containing protein. Cyclin B1 is associated with p34cdc2 in the cytoplasm. Thus we propose that the different localization of cyclin A and cyclin B1 in the cell cycle could be the means by which the two types of mitotic cyclin confer substrate specificity upon their associated PSTAIRE-containing protein kinase subunit..
Nevins, J.R.
Chellappan, S.P.
Mudryj, M.
Hiebert, S.
Devoto, S.
Horowitz, J.
Hunter, T.
Pines, J.
(1991). E2F Transcription Factor Is a Target for the RB Protein and the Cyclin A Protein. Cold spring harbor symposia on quantitative biology,
Vol.56
(0),
pp. 157-162.
PINES, J.
(1991). CYCLINS - WHEELS WITHIN WHEELS. Cell growth & differentiation,
Vol.2
(6),
pp. 305-310.
Mudryj, M.
Devoto, S.H.
Hiebert, S.W.
Hunter, T.
Pines, J.
Nevins, J.R.
(1991). Cell cycle regulation of the E2F transcription factor involves an interaction with cyclin A. Cell,
Vol.65
(7),
pp. 1243-1253.
Nishitani, H.
Ohtsubo, M.
Yamashita, K.
Iida, H.
Pines, J.
Yasudo, H.
Shibata, Y.
Hunter, T.
Nishimoto, T.
(1991). Loss of RCC1, a nuclear DNA-binding protein, uncouples the completion of DNA replication from the activation of cdc2 protein kinase and mitosis. The embo journal,
Vol.10
(6),
pp. 1555-1564.
Hunter, T.
Pines, J.
(1991). Cyclins and cancer. Cell,
Vol.66
(6),
pp. 1071-1074.
Pines, J.
Hunter, T.
(1991). Cyclin-dependent kinases: a new cell cycle motif?. Trends in cell biology,
Vol.1
(5),
pp. 117-121.
Weber, M.
Kubiak, J.Z.
Arlinghaus, R.B.
Pines, J.
Maro, B.
(1991). c-mos proto-oncogene product is partly degraded after release from meiotic arrest and persists during interphase in mouse zygotes. Developmental biology,
Vol.148
(1),
pp. 393-397.
PINES, J.
HUNTER, T.
(1991). HUMAN CELL-DIVISION - THE INVOLVEMENT OF CYCLINS-A AND CYCLINS-B1, AND MULTIPLE CDC2S. Cold spring harbor symposia on quantitative biology,
Vol.56,
pp. 449-463.
NEVINS, J.R.
CHELLAPPAN, S.P.
MUDRYJ, M.
HIEBERT, S.
DEVOTO, S.
HOROWITZ, J.
HUNTER, T.
PINES, J.
(1991). E2F TRANSCRIPTION FACTOR IS A TARGET FOR THE RB PROTEIN AND THE CYCLIN-A PROTEIN. Cold spring harbor symposia on quantitative biology,
Vol.56,
pp. 157-162.
Pines, J.
Hunter, T.
(1990). p34cdc2: the S and M kinase?. The new biologist,
Vol.2
(5),
pp. 389-401.
show abstract
In the yeast cell cycle, the induction of two very different processes, DNA synthesis (S-phase) and mitosis (M-phase), requires the same serine/threonine-specific protein kinase p34cdc2, which has been highly conserved through evolution. On the basis of work conducted largely in multicellular eukaryotes, it has recently been suggested that p34cdc2 is able to perform these two mutually exclusive roles by phosphorylating different sets of substrates through a cell cycle-dependent association with other proteins that dictate the substrate specificity of the protein kinase. To recognize its mitotic substrates, p34cdc2 associates with one of the cyclins--a family of proteins of two distinct but related types (A and B) characterized by their periodic destruction at each mitosis. In interphase, the formation of a complex between p34cdc2 and another protein (or proteins) would allow the phosphorylation of a different set of proteins involved in the G1 to S transition. This review focuses on the evidence for this appealing simple model and the nature of the putative substrates proposed..
Pines, J.
Hunter, T.
(1990). Human cyclin A is adenovirus E1A-associated protein p60 and behaves differently from cyclin B. Nature,
Vol.346
(6286),
pp. 760-763.
Yamashita, K.
Yasuda, H.
Pines, J.
Yasumoto, K.
Nishitani, H.
Ohtsubo, M.
Hunter, T.
Sugimura, T.
Nishimoto, T.
(1990). Okadaic acid, a potent inhibitor of type 1 and type 2A protein phosphatases, activates cdc2/H1 kinase and transiently induces a premature mitosis-like state in BHK21 cells. The embo journal,
Vol.9
(13),
pp. 4331-4338.
MINSHULL, J.
PINES, J.O.
GOLSTEYN, R.O.
STANDART, N.
MACKIE, S.
COLMAN, A.
BLOW, J.
RUDERMAN, J.V.
WU, M.
HUNT, T.I.
(1989). The role of cyclin synthesis, modification and destruction in the control of cell division. Journal of cell science,
Vol.1989
(Supplement_12),
pp. 77-97.
show abstract
Summary
This paper reviews our current knowledge of the cyclins based on observations of the oocytes and eggs of sea urchins, clams and frogs. Cyclins are proteins found in all eukaryotes whose special property is rapid destruction at specific stages in the cell cycle. The cyclins fall into three families. A-type cyclins have been found in clams, flies and frogs. B-type cyclins have been found in clams, flies, frogs, sea urchins and fission yeast. A more distantly related family of three genes is found in Saccharomyces cerevisiae. B-type cyclins appear to be required for cells to enter mitosis, and their destruction is thought to be necessary for exit from mitosis. We describe evidence in support of these ideas, and describe various conditions under which cyclin destruction is delayed or deranged. We conclude with a discussion of the relationship between the cyclins and maturation- (or M phase-) promoting factor and some ideas on how the cyclins may work..
Meijer, L.
Arion, D.
Golsteyn, R.
Pines, J.
Brizuela, L.
Hunt, T.
Beach, D.
(1989). Cyclin is a component of the sea urchin egg M-phase specific histone H1 kinase. The embo journal,
Vol.8
(8),
pp. 2275-2282.
Pines, J.
Hunter, T.
(1989). Isolation of a human cyclin cDNA: Evidence for cyclin mRNA and protein regulation in the cell cycle and for interaction with p34cdc2. Cell,
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(5),
pp. 833-846.
Felix, M.A.
Pines, J.
Hunt, T.
Karsenti, E.
(1989). A post-ribosomal supernatant from activated Xenopus eggs that displays post-translationally regulated oscillation of its cdc2+ mitotic kinase activity. The embo journal,
Vol.8
(10),
pp. 3059-3069.
Pines, J.
Hunt, T.
(1987). Molecular cloning and characterization of the mRNA for cyclin from sea urchin eggs. The embo journal,
Vol.6
(10),
pp. 2987-2995.
Standart, N.
Minshull, J.
Pines, J.
Hunt, T.
(1987). Cyclin synthesis, modification and destruction during meiotic maturation of the starfish oocyte. Developmental biology,
Vol.124
(1),
pp. 248-258.
Zernicka-Goetz, M.
Pines, J.
Cell Lineage Analysis: Applications of Green Fluorescent Protein. ,
,
pp. 279-287.
Atkin, J.
Halova, L.
Ferguson, J.
Hitchin, J.R.
Lichawska-Cieslar, A.
Jordan, A.M.
Pines, J.
Wellbrock, C.
Petersen, J.
Torin1 mediated TOR kinase inhibition reduces Wee1 levels and advances mitotic commitment in fission yeast and HeLa cells. Journal of cell science,
.
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The Target Of Rapamycin TOR kinase regulates cell growth and division. Rapamycin only inhibits a subset of TOR activities. Here we show that in contrast to the mild impact of rapamycin on cell division, blocking the catalytic site of TOR with the Torin1 inhibitor completely arrests growth without cell death in S.pombe. A mutation of the Tor2 TORC1 glycine residue (G2040D) that lies adjacent to the key Torin interacting tryptophan provides Torin1 resistance, confirming Torin1's specificity for TOR. Using this mutation we show that Torin1 advanced mitotic onset before inducing growth arrest. In contrast to TOR inhibition with Rapamycin, regulation by either Wee1 or Cdc25 was sufficient for this Torin1 induced advanced mitosis. Torin1 promoted a Polo and Cdr2 kinase controlled drop in Wee1 levels. Experiments in human cell lines re-capitulated these yeast observations; mTOR was inhibited by Torin1, Wee1 levels declined and mitotic commitment was advanced in HeLa cells. Thus, the regulation of the mitotic inhibitor Wee1 by TOR signalling is a conserved mechanism that helps to couple cell cycle and growth controls..
Sedgwick, G.G.
Hayward, D.G.
Di Fiore, B.
Pardo, M.
Yu, L.
Pines, J.
Nilsson, J.
Mechanisms controlling the temporal degradation of Nek2A and Kif18A by the APC/C–Cdc20 complex. The embo journal,
Vol.32
(2),
pp. 303-314.
Florindo, C.
Perdigão, J.
Fesquet, D.
Schiebel, E.
Pines, J.
Tavares, Á.A.
Human Mob1 proteins are required for cytokinesis by controlling microtubule stability. Journal of cell science,
.
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The completion of cytokinesis requires abscission of the midbody, a microtubule-rich cytoplasmic bridge that connects the daughter cells before their final separation. Although it has been established that both the midbody structure and membrane fusion are essential for abscission, the biochemical machinery and the cellular processes of abscission remain ill-defined. Here we report that human Mob1A and Mob1B proteins are involved in the regulation of abscission of the intercellular bridge. The Mob family is a group of highly conserved proteins in eukaryotes, described as binding partners as well as co-activators of protein kinases of the Ndr family, and as members of the Hippo pathway. We show that depletion of Mob1A and Mob1B by RNAi causes abscission failure as a consequence of hyper-stabilization of microtubules in the midbody region. Interestingly, depleting Mob1 also increases cell motility after cytokinesis, and induces prolonged centriole separation in G1 phase. In contrast, centrosomes fail to split when either Mob1A or Mob1B is overexpressed. Our findings indicate that human Mob1 proteins are involved in the regulation of microtubule stability at the midbody. We conclude that Mob1A and Mob1B are needed for cell abscission and centriole re-joining after telophase and cytokinesis..