The PMK-1 pathway regulated by stress response. (A,B) Activation of PMK-1 by oxidative stress. Animals were treated with oxidative stress agents. Extracts prepared from each animal were immunoblotted (IB) with anti-phospho p38 and anti-PMK-1. (t-BOOH) t-butyl peroxide. (C,D) Stress sensitivity in mutants. (C) Animals were scored for survival following exposure to sodium arsenite (5 mM) in M9 buffer for the indicated times. (D) Wild-type (N2) and sek-1 mutant animals harboring control vector or transgene as an extrachromosomal array were scored for survival after they had been placed in M9 containing sodium arsenite (5 mM) for 24 h.

Stress-induced gcs-1 expression and SKN-1 localization. (A,B) Effect of the PMK-1 pathway on stress-induced gcs-1 expression. Animals harboring the Pgcs-1::gfp transgene as an extrachromosomal array were treated with sodium arsenite (5 mM) for 1 h. These animals were then transferred to NGM plates and incubated for 3 h. Nomarski (DIC) and fluorescent (GFP) views are shown in A. (B) Percentages of animals in each expression category are listed. Low refers to animals in which intestinal GFP was present at high levels anteriorly and posteriorly, but was not detected in between. High indicates that a GFP was present at high levels throughout most of the intestine. (C,D) Effect of the PMK-1 pathway on stress-induced SKN-1 localization. Animals integrated with the skn-1::gfp transgene were treated with sodium arsenite (5 mM) for 1 h. Fluorescent views are shown in C. Accumulation of SKN-1::GFP in intestinal cell nuclei is indicated by arrows. (D) Percentages of animals in each expression category are listed. Low refers to animals in which SKN-1::GFP was barely detectable in intestinal nuclei. High indicates that a strong SKN-1::GFP signal was present in all intestinal nuclei.

Phosphorylation of SKN-1. (A,C) Phosphorylation of SKN-1 by PMK-1. HEK293 cells were transfected with control vector (-), HA-PMK-1, HA-PMK-1(K73R), and Flag-SEK-1 as indicated. Complexes immunoprecipitated with anti-HA were used for in vitro kinase reactions with GST–SKN-1. The amounts of immunoprecipitated HA-PMK-1 were determined with anti-HA. Expression of Flag-SEK-1 was determined by immunoblotting with anti-Flag in whole-cell extracts (WCEs). (B) Amino acid sequence of SKN-1 containing the putative MAPK phosphorylation sites. The phosphorylation sites are indicated by asterisks. (CNC) Cap-N-Collar domain; (BR) basic region.

Effects of SKN-1 phosphorylation. (A) Effect of the skn-1(S74, 340A) mutation on arsenate sensitivity. skn-1(zu67) mutant animals with control vector or transgene as an extrachromosomal array were scored for survival after they had been placed in M9 containing sodium arsenate (3 mM) for 24 h. (B,C) Effect of the skn-1 mutation on stress-induced SKN-1 localization. Wild-type (N2) or sek-1 mutant animals harboring the skn-1::gfp transgene as an extrachromosomal array were treated with sodium arsenate (5 mM) for 1 h. Fluorescent views are shown in B. Accumulation of SKN-1::GFP in intestinal cell nuclei is indicated by arrows. (C) Percentages of animals in each expression category are listed.

The p38 MAPK pathways in C. elegans. See text for details.