Intrinsically active Hog1 proteins as well as Hog1^WT are autophosphorylated in response to osmostress in the absence of Pbs2.

<p>A. Phosphorylation levels of wild type Hog1 (Hog1^WT) and of the intrinsically active Hog1 proteins, Hog1^D170A and Hog1^F318L, were tested when expressed in <i>hog1</i>Δ<i>pbs2</i>Δ cells (lanes 3–8) or in <i>hog1</i>Δ cells (lanes 9–14), before (−) and 10 minutes after (+) exposure to 1 M NaCl. <i>hog1</i>Δ<i>pbs2</i>Δ cells harboring an empty vector (lanes 1–2) served as a negative control. Cultures were grown on YNB(-URA). B. Endogenous, native Hog1^WT protein is phosphorylated in <i>pbs2</i>Δ cells. Phosphorylation levels of Hog1^WT were tested in cells of the YPH102 (WT), <i>hog1</i>Δ and <i>pbs2</i>Δ strains, grown on YPD (−NaCl) and 10 minutes after transfer to YPD media supplemented with 1 M NaCl (+NaCl). C. Kinase-dead Hog1 is not phosphorylated in <i>hog1</i>Δ<i>pbs2</i>Δ cells. Phosphorylation levels of wild type Hog1 (Hog1^WT) and kinase-dead Hog1 (Hog1^K52A) were tested when expressed in <i>hog1</i>Δ strain (lanes 1–4) or <i>hog1</i>Δ<i>pbs2</i>Δ strain (lanes 5–8), before (−) and 10 minutes after (+) exposure to 1 M NaCl.