scholarly journals cdc2 kinase-induced destabilization of MAP2-coated microtubules in Xenopus egg extracts

1992 ◽  
Vol 101 (1) ◽  
pp. 69-78 ◽  
Author(s):  
S. Faruki ◽  
M. Doree ◽  
E. Karsenti
Keyword(s):  
Steady State ◽  
Bovine Brain ◽  
Microtubule Dynamics ◽  
Cdc2 Kinase ◽  
Stabilizing Effect ◽  
Egg Extracts ◽  
Phosphorylation Event ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts ◽  
Stabilizing Factors

During the interphase to metaphase transition, microtubules are destabilized by a cdc2 kinase-dependent phosphorylation event. This destabilization is due to a dramatic increase in the rate at which each growing microtubule starts to shrink (catastrophe rate). In principle, this could be brought about by lowering the affinity of stabilizing MAPs for the microtubule wall, by activating a factor that would actively increase the catastrophe rate or by an alteration of both parameters. Here we examine the stabilizing effect of bovine brain MAP2 on microtubules assembled in interphase Xenopus egg extracts. We show that this MAP strongly stabilizes microtubules assembled in the extracts against nocodazole-induced depolymerization. However, it does not protect them from the cdc2 kinase-induced shortening and destabilization. Moreover, the steady-state length of centrosome-nucleated microtubules in cdc2-treated extracts containing MAP2 is similar to that found in extracts lacking exogenous MAP2. We also show that although exogenous MAP2 is phosphorylated by cdc2 kinase in the extract, this is not the cause of microtubule destabilization. These results indicate that increased microtubule dynamics during mitosis is due to the activation of a factor that can function independently of the presence of active, stabilizing factors.

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 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 Phosphorylation and activation of the Xenopus Cdc25 phosphatase in the absence of Cdc2 and Cdk2 kinase activity.

1995 ◽  
Vol 6 (2) ◽  
pp. 215-226 ◽  
Author(s):  
T Izumi ◽  
J L Maller
Keyword(s):  
Kinase Activity ◽  
Cdc2 Kinase ◽  
Nuclear Envelope Breakdown ◽  
Dual Specificity ◽  
M Phase ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

The M-phase inducer, Cdc25C, is a dual-specificity phosphatase that directly phosphorylates and activates the cyclin B/Cdc2 kinase complex, leading to initiation of mitosis. Cdc25 itself is activated at the G2/M transition by phosphorylation on serine and threonine residues. Previously, it was demonstrated that Cdc2 kinase is capable of phosphorylating and activating Cdc25, suggesting the existence of a positive feedback loop. In the present study, kinases other than Cdc2 that can phosphorylate and activate Cdc25 were investigated. Cdc25 was found to be phosphorylated and activated by cyclin A/Cdk2 and cyclin E/Cdk2 in vitro. However, in interphase Xenopus egg extracts with no detectable Cdc2 and Cdk2, treatment with the phosphatase inhibitor microcystin activated a distinct kinase that could phosphorylate and activate Cdc25. Microcystin also induced other mitotic phenomena such as chromosome condensation and nuclear envelope breakdown in extracts containing less than 5% of the mitotic level of Cdc2 kinase activity. These findings implicate a kinase other than Cdc2 and Cdk2 that may initially activate Cdc25 in vivo and suggest that this kinase may also phosphorylate M-phase substrates even in the absence of Cdc2 kinase.

scholarly journals Control of microtubule dynamics by the antagonistic activities of XMAP215 and XKCM1 in Xenopus egg extracts

10.1038/71330 ◽  
1999 ◽  
Vol 2 (1) ◽  
pp. 13-19 ◽  
Author(s):  
Régis Tournebize ◽  
Andrei Popov ◽  
Kazuhisa Kinoshita ◽  
Anthony J. Ashford ◽  
Sonja Rybina ◽  
...  
Keyword(s):  
Microtubule Dynamics ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

Purification of recombinant cyclin B1/cdc2 kinase from Xenopus egg extracts

1997 ◽  
pp. 12-28 ◽  
Author(s):  
Heike Wilhelm ◽  
Søren S.L. Andersen ◽  
Eric Karsenti
Keyword(s):  
Cyclin B1 ◽  
Cdc2 Kinase ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts
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