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. 2009 Dec 28;285(9):6761–6769. doi: 10.1074/jbc.M109.093237

FIGURE 3.

FIGURE 3.

Mutation of serine residues near the L483 site increases the steady-state levels of Wee1. A, sequence of the region of human Wee1 identified as required for turnover. Leucine 483 and serine 472 are underlined. B, human Wee1 is phosphorylated at serine 472. FLAG-tagged Wee1 was immunostained from transfected 293T cells, and the resulting immunoprecipitate is resolved by SDS-PAGE. The bands were excised, and liquid chromatography-MS/MS was performed on Wee1 band after trypsin digest. Scan indicated +80 AMU on serine 472, suggesting that it was phosphorylated C. Mutation of activation domain residues 471, 472, or 483 stabilizes Wee1. HeLa cells were transfected with either wild-type (WT) Wee1 or the indicated mutants. The known sites required for Wee1 destruction are serines 53 and 123. C, pulse-chase analysis of Myc-wild-type Wee1, Myc-S53A/123A-Wee1, or Myc-S472A/L483F. A representative assay is shown.