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. 2013 Jan 31;9(1):e1003262. doi: 10.1371/journal.pgen.1003262

Figure 3. Dot1 contributes to Mek1 activation by autophosphorylation.

Figure 3

(A) Whole cell extracts (WCE) from a zip1 ndt80 culture at 24 h in meiosis were incubated in the presence (+) or absence (−) of lambda phosphatase (λPPase). (B), (C) and (D) Detection of different phosphorylated forms of Mek1 in ndt80-arrested cells after 24 h in meiosis using high-resolution Phos-tag gels. Basal Mek1 (line) and several phosphorylated forms (black and white arrowheads) are indicated; see text for explanation. PGK or Ponceau S staining were used as loading controls. Asterisk in (D) marks a weak non-specific band. (E) Schematic representation of a model for the sequential phosphorylation events leading to Mek1 activation and the relevant mutations analyzed above. (1) Priming phosphorylation by Mec1/Tel1 (black arrowhead in B, C, D) is followed by (2) autophosphorylation of Mek1 (white arrowheads in B, C, D) leading to its full activation and (3) the checkpoint response. H3K79 methylation by Dot1 contributes to Mek1 autophosphorylation. Strains were: (A); DP428 (zip1). (B); DP428 (zip1), DP701 (zip1 hop1) and DP655 (zip1 dot1). (C); DP428 (zip1), DP655 (zip1 dot1), DP680 (zip1 mec1), DP861 (zip1 mec1 tel1), DP877 (zip1 rad24 tel1), DP728 (zip1 spo11) and DP674 (zip1 mek1Δ). (D); DP885 (zip1), DP890 (zip1 dot1), DP886 (zip1 mek1-T327A), DP887 (zip1 mek1-T331A), DP888 (zip1 mek1-K199R), DP674 (zip1 mek1Δ), DP680 (zip1 mec1) and DP861 (zip1 mec1 tel1).