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14-3-3 binding regulates catalytic activity of human Wee1 kinase
C J Rothblum-Oviatt1, C E Ryan, H Piwnica-Worms
1Department of Cell Biology and Physiology, Howard Hughes Medical Institute, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, Missouri 63110, USA.
Abstract:
The mitotic inducer Cdc2 is negatively regulated, in part, by phosphorylation on tyrosine 15. Human Wee1 is a tyrosine-specific protein kinase that phosphorylates Cdc2 on tyrosine 15. Human Wee1 is subject to multiple levels of regulation including reversible phosphorylation, proteolysis, and protein-protein interactions. Here we have investigated the contributions made by 14-3-3 binding to human Wee1 regulation and function. We report that the interactions of 14-3-3 proteins with human Wee1 are reduced during mitosis and are stable in the presence of the protein kinase inhibitor UCN-01. A mutant of Wee1 that is incapable of binding to 14-3-3 proteins has lower enzymatic activity, and this likely accounts for its reduced potency relative to wild-type Wee1 in inducing a G(2) cell cycle delay when overproduced in vivo. These findings indicate that 14-3-3 proteins function as positive regulators of the human Wee1 protein kinase.
Insights
14-3-3 proteins positively regulate human Wee1, a key kinase in cell cycle control. Reduced binding during mitosis and impaired function of non-binding Wee1 mutants highlight 14-3-3's crucial role.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- The cell cycle is tightly regulated by protein kinases, including Cdc2.
- Wee1 kinase phosphorylates Cdc2 at tyrosine 15, inhibiting its activity and inducing cell cycle arrest.
- Human Wee1 is regulated by phosphorylation, proteolysis, and protein interactions.
Purpose of the Study:
- To investigate the role of 14-3-3 protein interactions in regulating human Wee1 kinase activity and function.
- To determine how 14-3-3 binding to Wee1 changes during the cell cycle.
Main Methods:
- Studied 14-3-3 binding to human Wee1 using biochemical assays.
- Utilized a Wee1 mutant unable to bind 14-3-3 proteins.
- Assessed enzymatic activity and cell cycle delay (G2) in vivo.
Main Results:
- 14-3-3 binding to human Wee1 decreases during mitosis.
- 14-3-3 binding remains stable when treated with the protein kinase inhibitor UCN-01.
- Wee1 mutants lacking 14-3-3 binding exhibit reduced enzymatic activity and potency in inducing G2 delay.
Conclusions:
- 14-3-3 proteins act as positive regulators of human Wee1 kinase activity.
- 14-3-3 binding is essential for Wee1's function in cell cycle progression.