scholarly journals Activation of the p42 Mitogen-activated Protein Kinase Pathway Inhibits Cdc2 Activation and Entry into M-Phase in CyclingXenopus Egg Extracts

1998 ◽  
Vol 9 (2) ◽  
pp. 451-467 ◽  
Author(s):  
John C. Bitangcol ◽  
Andrew S.-S. Chau ◽  
Ellamae Stadnick ◽  
Manfred J. Lohka ◽  
Bryan Dicken ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Specific Inhibitor ◽  
Mapk Signaling ◽  
Cyclin B ◽  
Mitogen Activated Protein Kinase ◽  
M Phase ◽  
Egg Extracts ◽  
Mitogen Activated Protein ◽  
Xenopus Egg Extracts

We have added constitutively active MAP kinase/ERK kinase (MEK), an activator of the mitogen-activated protein kinase (MAPK) signaling pathway, to cycling Xenopus egg extracts at various times during the cell cycle. p42MAPK activation during entry into M-phase arrested the cell cycle in metaphase, as has been shown previously. Unexpectedly, p42MAPK activation during interphase inhibited entry into M-phase. In these interphase-arrested extracts, H1 kinase activity remained low, Cdc2 was tyrosine phosphorylated, and nuclei continued to enlarge. The interphase arrest was overcome by recombinant cyclin B. In other experiments, p42MAPK activation by MEK or by Mos inhibited Cdc2 activation by cyclin B. PD098059, a specific inhibitor of MEK, blocked the effects of MEK(QP) and Mos. Mos-induced activation of p42MAPK did not inhibit DNA replication. These results indicate that, in addition to the established role of p42MAPK activation in M-phase arrest, the inappropriate activation of p42MAPK during interphase prevents normal entry into M-phase.

scholarly journals Roles of Greatwall Kinase in the Regulation of Cdc25 Phosphatase

2008 ◽  
Vol 19 (4) ◽  
pp. 1317-1327 ◽  
Author(s):  
Yong Zhao ◽  
Olivier Haccard ◽  
Ruoning Wang ◽  
Jiangtao Yu ◽  
Jian Kuang ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Xenopus Oocytes ◽  
Mitogen Activated Protein Kinase ◽  
Mitotic Entry ◽  
M Phase ◽  
Egg Extracts ◽  
Mitogen Activated Protein ◽  
Xenopus Egg ◽  
Greatwall Kinase ◽  
Xenopus Egg Extracts

We previously reported that immunodepletion of Greatwall kinase prevents Xenopus egg extracts from entering or maintaining M phase due to the accumulation of inhibitory phosphorylations on Thr14 and Tyr15 of Cdc2. M phase–promoting factor (MPF) in turn activates Greatwall, implying that Greatwall participates in an MPF autoregulatory loop. We show here that activated Greatwall both accelerates the mitotic G2/M transition in cycling egg extracts and induces meiotic maturation in G2-arrested Xenopus oocytes in the absence of progesterone. Activated Greatwall can induce phosphorylations of Cdc25 in the absence of the activity of Cdc2, Plx1 (Xenopus Polo-like kinase) or mitogen-activated protein kinase, or in the presence of an activator of protein kinase A that normally blocks mitotic entry. The effects of active Greatwall mimic in many respects those associated with addition of the phosphatase inhibitor okadaic acid (OA); moreover, OA allows cycling extracts to enter M phase in the absence of Greatwall. Taken together, these findings support a model in which Greatwall negatively regulates a crucial phosphatase that inhibits Cdc25 activation and M phase induction.

scholarly journals Mitogen-activated Protein Kinase Kinase 1-dependent Golgi Unlinking Occurs in G2 Phase and Promotes the G2/M Cell Cycle Transition

2007 ◽  
Vol 18 (2) ◽  
pp. 594-604 ◽  
Author(s):  
Timothy N. Feinstein ◽  
Adam D. Linstedt
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Specific Inhibitor ◽  
Cell Cycle Checkpoint ◽  
Mitogen Activated Protein Kinase ◽  
M Cell ◽  
M Phase ◽  
Mitogen Activated Protein ◽  
Cell Cycle Transition ◽  
Protein Kinase Kinase

Two controversies have emerged regarding the signaling pathways that regulate Golgi disassembly at the G2/M cell cycle transition. The first controversy concerns the role of mitogen-activated protein kinase activator mitogen-activated protein kinase kinase (MEK)1, and the second controversy concerns the participation of Golgi structure in a novel cell cycle “checkpoint.” A potential simultaneous resolution is suggested by the hypothesis that MEK1 triggers Golgi unlinking in late G2 to control G2/M kinetics. Here, we show that inhibition of MEK1 by RNA interference or by using the MEK1/2-specific inhibitor U0126 delayed the passage of synchronized HeLa cells into M phase. The MEK1 requirement for normal mitotic entry was abrogated if Golgi proteins were dispersed before M phase by treatment of cells with brefeldin A or if GRASP65, which links Golgi stacks into a ribbon network, was depleted. Imaging revealed that unlinking of the Golgi apparatus begins before M phase, is independent of cyclin-dependent kinase 1 activation, and requires MEK signaling. Furthermore, expression of the GRASP family member GRASP55 after alanine substitution of its MEK1-dependent mitotic phosphorylation sites inhibited both late G2 Golgi unlinking and the G2/M transition. Thus, MEK1 plays an in vivo role in Golgi reorganization, which regulates cell cycle progression.

scholarly journals Regulation of Cdc2/Cyclin B Activation in Xenopus Egg Extracts via Inhibitory Phosphorylation of Cdc25C Phosphatase by Ca2+/Calmodium-dependent Kinase II

2003 ◽  
Vol 14 (10) ◽  
pp. 4003-4014 ◽  
Author(s):  
James R. A. Hutchins ◽  
Dina Dikovskaya ◽  
Paul R. Clarke
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Kinase Activity ◽  
Embryonic Cell ◽  
Cyclin B ◽  
Checkpoint Kinases ◽  
M Phase ◽  
Egg Extracts ◽  
Inhibitory Site

Activation of Cdc2/cyclin B kinase and entry into mitosis requires dephosphorylation of inhibitory sites on Cdc2 by Cdc25 phosphatase. In vertebrates, Cdc25C is inhibited by phosphorylation at a single site targeted by the checkpoint kinases Chk1 and Cds1/Chk2 in response to DNA damage or replication arrest. In Xenopus early embryos, the inhibitory site on Cdc25C (S287) is also phosphorylated by a distinct protein kinase that may determine the intrinsic timing of the cell cycle. We show that S287-kinase activity is repressed in extracts of unfertilized Xenopus eggs arrested in M phase but is rapidly stimulated upon release into interphase by addition of Ca2+, which mimics fertilization. S287-kinase activity is not dependent on cyclin B degradation or inactivation of Cdc2/cyclin B kinase, indicating a direct mechanism of activation by Ca2+. Indeed, inhibitor studies identify the predominant S287-kinase as Ca2+/calmodulin-dependent protein kinase II (CaMKII). CaMKII phosphorylates Cdc25C efficiently on S287 in vitro and, like Chk1, is inhibited by 7-hydroxystaurosporine (UCN-01) and debromohymenialdisine, compounds that abrogate G2 arrest in somatic cells. CaMKII delays Cdc2/cyclin B activation via phosphorylation of Cdc25C at S287 in egg extracts, indicating that this pathway regulates the timing of mitosis during the early embryonic cell cycle.

scholarly journals Katanin Is Responsible for the M-Phase Microtubule-severing Activity in Xenopus Eggs

1998 ◽  
Vol 9 (7) ◽  
pp. 1847-1861 ◽  
Author(s):  
Francis J. McNally ◽  
Susan Thomas
Keyword(s):  
Cell Cycle ◽  
Human Fibroblasts ◽  
Cyclin B ◽  
Microtubule Severing ◽  
Spindle Poles ◽  
M Phase ◽  
Egg Extracts ◽  
Dynamic Structures ◽  
Cycle Dependent ◽  
Xenopus Egg Extracts

Microtubules are dynamic structures whose proper rearrangement during the cell cycle is essential for the positioning of membranes during interphase and for chromosome segregation during mitosis. The previous discovery of a cyclin B/cdc2-activated microtubule-severing activity in M-phase Xenopus egg extracts suggested that a microtubule-severing protein might play an important role in cell cycle-dependent changes in microtubule dynamics and organization. However, the isolation of three different microtubule-severing proteins, p56, EF1α, and katanin, has only confused the issue because none of these proteins is directly activated by cyclin B/cdc2. Here we use immunodepletion with antibodies specific for a vertebrate katanin homologue to demonstrate that katanin is responsible for the majority of M-phase severing activity inXenopus eggs. This result suggests that katanin is responsible for changes in microtubules occurring at mitosis. Immunofluorescence analysis demonstrated that katanin is concentrated at a microtubule-dependent structure at mitotic spindle poles inXenopus A6 cells and in human fibroblasts, suggesting a specific role in microtubule disassembly at spindle poles. Surprisingly, katanin was also found in adult mouse brain, indicating that katanin may have other functions distinct from its mitotic role.

scholarly journals Inhibitor-2 induced M-phase arrest in Xenopus cycling egg extracts is dependent on MAPK activation

2011 ◽  
Vol 16 (4) ◽  
Author(s):  
Arian Khandani ◽  
Mahmood Mohtashami ◽  
Anne Camirand
Keyword(s):  
Cell Cycle ◽  
Mapk Pathway ◽  
Mitogen Activated Protein Kinase ◽  
Protein Phosphatase 1 ◽  
Mapk Activity ◽  
Phase Arrest ◽  
Evolutionarily Conserved ◽  
M Phase ◽  
Egg Extracts ◽  
Mitogen Activated Protein

AbstractThe evolutionarily-conserved protein phosphatase 1 (PP1) plays a central role in dephosphorylation of phosphoproteins during the M phase of the cell cycle. We demonstrate here that the PP1 inhibitor inhibitor-2 protein (Inh-2) induces an M-phase arrest in Xenopus cycling egg extracts. Interestingly, the characteristics of this M-phase arrest are similar to those of mitogen-activated protein kinase (p42MAPK)-induced M-phase arrest. This prompted us to investigate whether Inh-2-induced M-phase arrest was dependent on activation of the p42MAPK pathway. We demonstrate here that MAPK activity is required for Inh-2-induced M-phase arrest, as inhibition of MAPK by PD98059 allowed cycling extracts to exit M phase, despite the presence of Inh-2. We next investigated whether Inh-2 phosphorylation by the MAPK pathway was required to induce an M-phase arrest. We discovered that while p90Rsk (a MAPK protein required for M-phase arrest) is able to phosphorylate Inh-2, this phosphorylation is not required for Inh-2 function. Overall, our results suggest a novel mechanism linking p42MAPK and PP1 pathways during M phase of the cell cycle.

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