B-Raf and C-Raf are required for Ras-stimulated p42 MAP kinase activation in 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.

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A requirement for MAP kinase in the assembly and maintenance of the mitotic spindle

The Journal of Cell Biology ◽

10.1083/jcb.200304144 ◽

2003 ◽

Vol 161 (6) ◽

pp. 1021-1028 ◽

Cited By ~ 39

Author(s):

Melinda M. Horne ◽

Thomas M. Guadagno

Keyword(s):

Map Kinase ◽

Mitotic Spindle ◽

Kinase Inhibitor ◽

Map Kinase Phosphatase ◽

Egg Extracts ◽

Xenopus Egg ◽

Multiple Spindle ◽

Xenopus Egg Extracts ◽

Nih 3T3 ◽

Bipolar Spindles

Circumstantial evidence has suggested the possibility of microtubule-associated protein (MAP) kinase's involvement in spindle regulation. To test this directly, we asked whether MAP kinase was required for spindle assembly in Xenopus egg extracts. Either the inhibition or the depletion of endogenous p42 MAP kinase resulted in defective spindle structures resembling asters or half-spindles. Likewise, an increase in the length and polymerization of microtubules was measured in aster assays suggesting a role for MAP kinase in regulating microtubule dynamics. Consistent with this, treatment of extracts with either a specific MAP kinase kinase inhibitor or a MAP kinase phosphatase resulted in the rapid disassembly of bipolar spindles into large asters. Finally, we report that mitotic progression in the absence of MAP kinase signaling led to multiple spindle abnormalities in NIH 3T3 cells. We therefore propose that MAP kinase is a key regulator of the mitotic spindle.

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Investigating the determinants of mammalian Mastl kinase activation

10.1101/2020.06.30.179580 ◽

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.

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

Molecular Biology of the Cell ◽

10.1091/mbc.4.4.397 ◽

1993 ◽

Vol 4 (4) ◽

pp. 397-411 ◽

Cited By ~ 111

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.

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