Characterization of S. cerevisiae Hog1 protein activation upon bacterial LPS-exposure.
Joana M. Marques (1), Ricardo J. Rodrigues (2), Teresa Gonçalves (1)
(1) CNC, Inst.Microbiology, FMUC, Rua Larga, Coimbra, 3004-504, Portugal; (2) CNC, Inst. Biochemistry, FMUC, Rua Larga, Coimbra, 3004-504, Portugal
Yeast HOG MAPK pathway is generally related with the response to increases of the extracellular osmolarity, and Hog1p is the MAP kinase activated in order to achieve osmoadaptation. Some recent studies showed that this pathway is also activated with other stresses. Furthermore, the yeast Hog1p is homolog of the p38 mammalian cell protein kinase involved in the response to bacterial lipopolysaccharide (LPS). It is our aim to demonstrate that the functional homology observed between Hog1p and p38 is not restricted to osmoadaptation, but to other activators, particularly to LPS. In this study we present data showing that S. cerevisiae exposure to LPS triggers Hog1p phosphorylation. With a triple labelling immunocytochemical assay we show the presence of phosphorylated Hog1p in the nucleus, in a correlated-manner with its maximum phosphorylation point. The comparison of the phosphorylation kinetics of Hog1p under hiperosmotic conditions (0.8M NaCl) and upon LPS-exposure clearly showed that yeasts respond more slowly to the presence of LPS with a maximum phosphorylation at 3-6 h of incubation. Real-time RT-PCR analysis suggests that Hog1p is involved in the up-regulation of GPD1 mRNA levels upon LPS-exposure, which indicates that Hog1p is not only phosphorylated and translocated to the nucleus upon LPS-exposure, but also it is catalically active. Here we report for the first time that LPS triggers the phosphorylation/activation of the yeast homolog of mammalian p38, Hog1p.