Faculty Opinions recommendation of Greatwall kinase participates in the Cdc2 autoregulatory loop in Xenopus egg extracts.

2006 ◽  
Author(s):  
Iain Hagan
Keyword(s):  
Egg Extracts ◽  
Xenopus Egg ◽  
Greatwall Kinase ◽  
Xenopus Egg Extracts

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 Greatwall Kinase Participates in the Cdc2 Autoregulatory Loop in Xenopus Egg Extracts

2006 ◽  
Vol 22 (1) ◽  
pp. 83-91 ◽  
Author(s):  
Jiangtao Yu ◽  
Yong Zhao ◽  
ZeXiao Li ◽  
Simon Galas ◽  
Michael L. Goldberg
Keyword(s):  
Egg Extracts ◽  
Xenopus Egg ◽  
Greatwall Kinase ◽  
Xenopus Egg Extracts

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 The M Phase Kinase Greatwall (Gwl) Promotes Inactivation of PP2A/B55δ, a Phosphatase Directed Against CDK Phosphosites

2009 ◽  
Vol 20 (22) ◽  
pp. 4777-4789 ◽  
Author(s):  
Priscila V. Castilho ◽  
Byron C. Williams ◽  
Satoru Mochida ◽  
Yong Zhao ◽  
Michael L. Goldberg
Keyword(s):  
Cyclin B ◽  
Regulatory Subunit ◽  
Cyclin Dependent Kinases ◽  
M Phase ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Greatwall Kinase ◽  
Xenopus Egg Extracts ◽  
Phase Entry ◽  
Dependent Mechanism

We have previously shown that Greatwall kinase (Gwl) is required for M phase entry and maintenance in Xenopus egg extracts. Here, we demonstrate that Gwl plays a crucial role in a novel biochemical pathway that inactivates, specifically during M phase, “antimitotic” phosphatases directed against phosphorylations catalyzed by cyclin-dependent kinases (CDKs). A major component of this phosphatase activity is heterotrimeric PP2A containing the B55δ regulatory subunit. Gwl is activated during M phase by Cdk1/cyclin B (MPF), but once activated, Gwl promotes PP2A/B55δ inhibition with no further requirement for MPF. In the absence of Gwl, PP2A/B55δ remains active even when MPF levels are high. The removal of PP2A/B55δ corrects the inability of Gwl-depleted extracts to enter M phase. These findings support the hypothesis that M phase requires not only high levels of MPF function, but also the suppression, through a Gwl-dependent mechanism, of phosphatase(s) that would otherwise remove MPF-driven phosphorylations.

scholarly journals The bulk of unpolymerized actin in Xenopus egg extracts is ATP-bound.

1995 ◽  
Vol 6 (2) ◽  
pp. 227-236 ◽  
Author(s):  
J Rosenblatt ◽  
P Peluso ◽  
T J Mitchison
Keyword(s):  
Actin Polymerization ◽  
Critical Concentration ◽  
Large Fraction ◽  
Nucleotide Exchange ◽  
Chromatography Analysis ◽  
Thymosin Beta 4 ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

Non-muscle cells contain 15-500 microM actin, a large fraction of which is unpolymerized. Thus, the concentration of unpolymerized actin is well above the critical concentration for polymerization in vitro (0.2 microM). This fraction of actin could be prevented from polymerization by being ADP bound (therefore less favored to polymerize) or by being ATP bound and sequestered by a protein such as thymosin beta 4, or both. We isolated the unpolymerized actin from Xenopus egg extracts using immobilized DNase 1 and assayed the bound nucleotide. High-pressure liquid chromatography analysis showed that the bulk of soluble actin is ATP bound. Analysis of actin-bound nucleotide exchange rates suggested the existence of two pools of unpolymerized actin, one of which exchanges nucleotide relatively rapidly and another that apparently does not exchange. Native gel electrophoresis of Xenopus egg extracts demonstrated that most of the soluble actin exists in complexes with other proteins, one of which might be thymosin beta 4. These results are consistent with actin polymerization being controlled by the sequestration and release of ATP-bound actin, and argue against nucleotide exchange playing a major role in regulating actin polymerization.

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