scholarly journals Dephosphorylation of cdc25-C by a type-2A protein phosphatase: specific regulation during the cell cycle in Xenopus egg extracts.

1993 ◽  
Vol 4 (4) ◽  
pp. 397-411 ◽  
Author(s):  
P R Clarke ◽  
I Hoffmann ◽  
G Draetta ◽  
E Karsenti
Keyword(s):  
Protein Kinase ◽  
Protein Phosphatase ◽  
Cyclin B ◽  
Specific Inhibition ◽  
Kinase Activation ◽  
Protein Kinase Activation ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts ◽  
Low Activity

We have examined the roles of type-1 (PP-1) and type-2A (PP-2A) protein-serine/threonine phosphatases in the mechanism of activation of p34cdc2/cyclin B protein kinase in Xenopus egg extracts. p34cdc2/cyclin B is prematurely activated in the extracts by inhibition of PP-2A by okadaic acid but not by specific inhibition of PP-1 by inhibitor-2. Activation of the kinase can be blocked by addition of the purified catalytic subunit of PP-2A at a twofold excess over the activity in the extract. The catalytic subunit of PP-1 can also block kinase activation, but very high levels of activity are required. Activation of p34cdc2/cyclin B protein kinase requires dephosphorylation of p34cdc2 on Tyr15. This reaction is catalysed by cdc25-C phosphatase that is itself activated by phosphorylation. We show that, in interphase extracts, inhibition of PP-2A by okadaic acid completely blocks cdc25-C dephosphorylation, whereas inhibition of PP-1 by specific inhibitors has no effect. This indicates that a type-2A protein phosphatase negatively regulates p34cdc2/cyclin B protein kinase activation primarily by maintaining cdc25-C phosphatase in a dephosphorylated, low activity state. In extracts containing active p34cdc2/cyclin B protein kinase, dephosphorylation of cdc25-C is inhibited, whereas the activity of PP-2A (and PP-1) towards other substrates is unaffected. We propose that this specific inhibition of cdc25-C dephosphorylation is part of a positive feedback loop that also involves direct phosphorylation and activation of cdc25-C by p34cdc2/cyclin B. Dephosphorylation of cdc25-C is also inhibited when cyclin A-dependent protein kinase is active, and this may explain the potentiation of p34cdc2/cyclin B protein kinase activation by cyclin A. In extracts supplemented with nuclei, the block on p34cdc2/cyclin B activation by unreplicated DNA is abolished when PP-2A is inhibited or when stably phosphorylated cdc25-C is added, but not when PP-1 is specifically inhibited. This suggests that unreplicated DNA inhibits p34cdc2/cyclin B activation by maintaining cdc25-C in a low activity, dephosphorylated state, probably by keeping the activity of a type-2A protein phosphatase towards cdc25-C at a high level.

scholarly journals MAP kinase does not inactivate, but rather prevents the cyclin degradation pathway from being turned on in Xenopus egg extracts

1996 ◽  
Vol 109 (1) ◽  
pp. 239-246 ◽  
Author(s):  
A. Abrieu ◽  
T. Lorca ◽  
J.C. Labbe ◽  
N. Morin ◽  
S. Keyse ◽  
...  
Keyword(s):  
Map Kinase ◽  
Degradation Pathway ◽  
Cdc2 Kinase ◽  
Vitro Assay ◽  
Kinase Activation ◽  
Egg Extracts ◽  
Dependent Protein ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

Unfertilized frog eggs arrest at the second meiotic metaphase, due to cytostatic activity of the c-mos proto-oncogene (CSF). MAP kinase has been proposed to mediate CSF activity in suppressing cyclin degradation. Using an in vitro assay to generate CSF activity, and recombinant CL 100 phosphatase to inactivate MAP kinase, we confirm that the c-mos proto-oncogene blocks cyclin degradation through MAP kinase activation. We further show that for MAP kinase to suppress cyclin degradation, it must be activated before cyclin B-cdc2 kinase has effectively promoted cyclin degradation. Thus MAP kinase does not inactivate, but rather prevents the cyclin degradation pathway from being turned on. Using a constitutively active mutant of Ca2+/calmodulin dependent protein kinase II, which mediates the effects of Ca2+ at fertilization, we further show that the kinase can activate cyclin degradation in the presence of both MPF and the c-mos proto-oncogene without inactivating MAP kinase.

scholarly journals Cell Cycle Regulation of the Replication Licensing System: Involvement of a Cdk-dependent Inhibitor

1997 ◽  
Vol 136 (1) ◽  
pp. 125-135 ◽  
Author(s):  
Hiro M. Mahbubani ◽  
James P.J. Chong ◽  
Stephane Chevalier ◽  
Pia Thömmes ◽  
J. Julian Blow
Keyword(s):  
Cell Cycle ◽  
Single Cell ◽  
Kinase Inhibitor ◽  
Cyclin B ◽  
Initiation Factor ◽  
Chromosomal Dna ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts ◽  
Cycle Regulation

The replication licensing factor (RLF) is an essential initiation factor that is involved in preventing re-replication of chromosomal DNA in a single cell cycle. In Xenopus egg extracts, it can be separated into two components: RLF-M, a complex of MCM/P1 polypeptides, and RLF-B, which is currently unpurified. In this paper we investigate variations in RLF activity throughout the cell cycle. Total RLF activity is low in metaphase, due to a lack of RLF-B activity and the presence of an RLF inhibitor. RLF-B is rapidly activated on exit from metaphase, and then declines during interphase. The RLF inhibitor present in metaphase extracts is dependent on the activity of cyclin-dependent kinases (Cdks). Affinity depletion of Cdks from metaphase extracts removed the RLF inhibitor, while Cdc2/cyclin B directly inhibited RLF activity. In metaphase extracts treated with the protein kinase inhibitor 6-dimethylaminopurine (6-DMAP), both cyclin B and the RLF inhibitor were stabilized although the extracts morphologically entered interphase. These results are consistent with studies in other organisms that invoke a key role for Cdks in preventing re-replication of DNA in a single cell cycle.

scholarly journals Control of microtubule dynamics and length by cyclin A- and cyclin B-dependent kinases in Xenopus egg extracts.

1992 ◽  
Vol 118 (5) ◽  
pp. 1097-1108 ◽  
Author(s):  
F Verde ◽  
M Dogterom ◽  
E Stelzer ◽  
E Karsenti ◽  
S Leibler
Keyword(s):  
Steady State ◽  
Fold Increase ◽  
Cyclin A ◽  
Microtubule Dynamics ◽  
Cyclin B ◽  
Free System ◽  
Egg Extracts ◽  
Dynamical Parameters ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

In eukaryotic cells, the onset of mitosis involves cyclin molecules which interact with proteins of the cdc2 family to produce active kinases. In vertebrate cells, cyclin A dependent kinases become active in S- and pro-phases, whereas a cyclin B-dependent kinase is mostly active in metaphase. It has recently been shown that, when added to Xenopus egg extracts, bacterially produced A- and B-type cyclins associate predominantly with the same kinase catalytic subunit, namely p34cdc2, and induce its histone H1 kinase activity with different kinetics. Here, we show that in the same cell free system, both the addition of cyclin A and cyclin B changes microtubule behavior. However, the cyclin A-dependent kinase does not induce a dramatic shortening of centrosome-nucleated microtubules whereas the cyclin B-dependent kinase does, as previously reported. Analysis of the parameters of microtubule dynamics by fluorescence video microscopy shows that the dramatic shortening induced by the cyclin B-dependent kinase is correlated with a several fold increase in catastrophe frequency, an effect not observed with the cyclin A-dependent kinase. Using a simple mathematical model, we show how the length distributions of centrosome-nucleated microtubules relate to the four parameters that describe microtubule dynamics. These four parameters define a threshold between unlimited microtubule growth and the establishment of steady-state dynamics, which implies that well defined steady-state length distributions can be produced by regulating precisely the respective values of the dynamical parameters. Moreover, the dynamical model predicts that increasing catastrophe frequency is more efficient than decreasing the rescue frequency to reduce the average steady state length of microtubules. These theoretical results are quantitatively confirmed by the experimental data.

scholarly journals Investigating the determinants of mammalian Mastl kinase activation

2020 ◽  
Author(s):  
Mehmet Erguven ◽  
M. Kasim Diril
Keyword(s):  
Expression System ◽  
Activation Loop ◽  
Middle Region ◽  
Kinase Activation ◽  
Viral Expression ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Greatwall Kinase ◽  
Xenopus Egg Extracts ◽  
Atypical Member

ABSTRACTMastl (Greatwall) kinase is an essential mitotic protein kinase. Mastl is an atypical member of AGC family with a unique long stretch of non-conserved middle region. The mechanism of its phosphorylation dependent activation has been studied in Xenopus egg extracts, revealing several phosphosites that were suggested to be crucial for kinase activation. These residues correspond to T193 and T206 in the activation loop, and S861 in the C-tail of mouse Mastl. By combining a chemically inducible knockout system to deplete the endogenous Mastl and a viral expression system to ectopically express the mutant variants, we obtained a viable knockout clone that expresses the S861A and S861D mutants. We observed that proliferation rates of the MastlS861A and MastlS861D clones were comparable. Our results have revealed that phosphorylation of the turn motif phosphosite (S861) is auxiliary and it is not indispensable for Mastl function.

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.

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