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Multisite M-Phase Phosphorylation of Xenopus Wee1A

Department of Molecular Pharmacology, Stanford University School of Medicine, Stanford, California 94305-5174,¹ Center for Cell Signaling Research, Division of Molecular Life Sciences and College of Pharmacy, Ewha Womans University, Seoul 120-750, South Korea²

Received 3 August 2005/ Returned for modification 6 September 2005/ Accepted 12 September 2005

The Cdk1 inhibitor Wee1 is inactivated during mitotic entry by proteolysis, translational regulation, and transcriptional regulation. Wee1 is also regulated by posttranslational modifications, and here we have identified five phosphorylation sites in the N-terminal domain of embryonic Xenopus Wee1A through a combination of mutagenesis studies and matrix-assisted laser desorption ionization-time of flight mass spectrometry. All five sites conform to the Ser-Pro/Thr-Pro consensus for proline-directed kinases like Cdks. Three of the sites (Ser 38, Thr 53, and Ser 62) are required for the mitotic gel shift, and at least two of these sites (Ser 38 and Thr 53) regulate the proteolysis of Wee1A during interphase. The other two sites (Thr 104 and Thr 150) are primarily responsible for the mitotic inactivation of Wee1A. Alanine mutants of Thr 150 or Thr 104 had an increased capacity to inhibit mitotic entry in cyclin B-treated interphase extracts, and Thr 150 was found to be transiently phosphorylated just prior to nuclear envelope breakdown in cycling egg extracts. These findings establish the phosphorylation-dependent direct inactivation of Wee1A as a critical mechanism for the promotion of M-phase entry. These results also show that multisite phosphorylation cooperatively inactivates Wee1A and cooperatively promotes Wee1A proteolysis.

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• Okamoto, K., Sagata, N. (2007). Mechanism for inactivation of the mitotic inhibitory kinase Wee1 at M phase. Proc. Natl. Acad. Sci. USA 104: 3753-3758 [Abstract] [Full Text]