Fig. 4
A kinase screen identified several kinases that phosphorylate the N terminus of Mrc1. a Six kinases phosphorylate the N terminus of Mrc1. In the screen, 123 tagged-purified yeast kinases were incubated in an in vitro kinase assay with a fragment (1–360 aa) of Mrc1 or its corresponding mutant in which the three phosphorylation sites Thr169, Ser215, and Ser229 were mutated to alanine (mrc13A) (see “Methods” section). Out of these 123 in vivo-purified kinases, 25 kinases (in blue) phosphorylated GST–Mrc1 in an in vitro kinase assay. Six of these 25 kinases phosphorylated GST–Mrc1 but not GST-mrc13A. b–f Mpk1 kinase mediates the Mrc1-dependent cell cycle delay upon heat stress. b Mpk1 is phosphorylated in vivo upon heat stress. Synchronized cells were released into S phase and subjected to heat stress. Mpk1 phosphorylation over time was followed by western blotting using specific antibodies. Nonphosphorylated Mpk1 was blotted as a loading control (c) Mrc1 is phosphorylated in vivo by Mpk1 upon heat stress. This experiment was carried out as in Fig. 1a and was assayed as in b. d Mrc1 and Mpk1 interact in vivo. Mrc1–TAP was immunoprecipitated (IP) from cells and coprecipitated Mpk1-myc was assayed using western blotting. e mpk1 cells bypass the DNA replication delay caused by heat stress. Pheromone presynchronized cells were arrested at S-phase onset using the cdc7AID (auxin-induced degron) system before their release at 37 °C (heat stress). DNA replication over time was followed using FACS. f mpk1 cells display a higher percentage of Rad52-YFP foci upon heat stress. Data represent the mean and standard deviation of three independent experiments. Asterisks indicate statistically significant differences by Student's t test (*p < 0.05, **p < 0.01, ***p < 0.005) of stress versus control conditions
