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. 2010 Dec 1;9(23):4688–4702. doi: 10.4161/cc.9.23.14050

Figure 5.

Figure 5

Mek1 and/or Cds1 phosphorylate the T218, T275 and/or T422 residues of S. pombe Mus81 in vitro. (A) Schematic diagram of GST-Mus81 fragments. Mus81 fragment amino acid numbers are shown left of each box. (B ∼ E) refers to fragments whose in vitro kinase assay data are shown below in (B ∼ E). Black and gray boxes indicate phosphorylated and non-phosphorylated fragments, respectively. Sub1 (substrate 1) and Sub2 (substrate 2) signify the GST-Mus81 fragments that were used for the kinase assay in (B). (B–F) SDS-PAGE parts for in vitro kinase reactions performed with truncated GST-Mus81 fragments and Mek1-GFP (i) or Cds1-2HA (ii) as kinases. Simply Blue staining (SB, left parts, C–F) shows a loading control. (B) The truncated GST-fusion proteins used were those containing Sub1 and Sub2, and derivatives with replacement of the T218 and T422 residues by alanine (A). (C) The truncated GST-fusion proteins used were those containing amino acids 1–127, 32–217, 219–421 and 423–608 of Mus81. An asterisk shows the autophosphorylated band of Mek1 kinase. (D) The truncated GST-fusion proteins used were those containing amino acids 219–421, 219–281, 252–362 and 336–421 of Mus81. (E) The truncated GST-fusion proteins used were those containing amino acids 252–362, 252–313, 287–362 and 317–362 of Mus81. (F) The truncated GST-fusion proteins used were those containing amino acids 252–281 and 276–313 of Mus81, and derivatives with replacement of T260, S270 and T275 residues by (A). GST-252-281 (3A) signifies that this GST-Mus81 fragment harbors all of these replacements.