Multisite M-phase phosphorylation of Xenopus Wee1A.
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.
Kim
Sun Young
SY
Department of Molecular Pharmacology, Stanford University School of Medicine, Stanford, CA 94305-5174, USA.
Song
Eun Joo
EJ
Lee
Kong-Joo
KJ
Ferrell
James E
JE
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002455:Cell Division; D016213:Cyclins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007399:Interphase; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D010769:Phosphothreonine / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D029867:Xenopus Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis / Q000235:genetics / Q000378:metabolism*
D000906:Antibodies; D018797:Cell Cycle Proteins; D016213:Cyclins; D009687:Nuclear Proteins; D029867:Xenopus Proteins; D010769:Phosphothreonine; C530005:WEE1 protein, Xenopus; D011505:Protein-Tyrosine Kinases