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. 2009 Oct 5;29(24):6449–6461. doi: 10.1128/MCB.00794-09

FIG. 7.

FIG. 7.

Mpk1 undergoes serine phosphorylation in response to caffeine treatment. (A) Caffeine induces serine phosphorylation of Mpk1. Yeast strain DL456 (mpk1Δ), transformed with a plasmid expressing FLAG-tagged Mpk1 (p2313), was either maintained at 25°C or subjected to cell wall stress (heat shock) by changing the temperature to 39°C (+) or by adding 8 mM caffeine (+) for 2 h. Cell extracts were subjected to immunoprecipitation of Mpk1-FLAG, followed by immunodetection of total Mpk1 with anti-FLAG antibodies, activated Mpk1 with phospho-p42/p44 antibodies (phospho-TEY), and serine phosphorylation of Mpk1 with antiphosphoserine antibodies. (B) Caffeine-induced serine phosphorylation of Mpk1 is independent of Mpk1 activation state or catalytic activity. Yeast strain DL456 (mpk1Δ), transformed with a plasmid expressing FLAG-tagged Mpk1 (p2313), nonactivatable Mpk1-TAYF (p2316), or catalytically inactive Mpk1-K54R (p2317), were subjected to caffeine stress (+) and processed as described above for detection of Mpk1 phosphorylated on serine and total Mpk1.