Article Figures & Data
Figures
- FIG. 1.
Heavy metal stress sensitivity in C. elegans mutants. (A) Schematic representations of the structures of human PAK1 and C. elegans PAK proteins. Striped boxes and gray boxes represent the CRIB motif and kinase domain, respectively. The percentages of amino acid similarity in the CRIB motif and kinase domain are shown above the boxes. The number of amino acids (a.a.) in the proteins are shown in parentheses to the right of the schematic representation. (B) Schematic representation of the structures of C. elegans PAK genes. Exons and introns are indicated by boxes and lines, respectively. Striped boxes and gray boxes show the CRIB motif and kinase domain, respectively. The thick lines underneath the schematic representation indicate the extent of the deleted region in each deletion mutant. The position of the cy2 mutation is indicated by an asterisk. (C to H) Copper sensitivity. Each animal was cultured from embryogenesis on NGM plates containing 100 μM copper ion (+) and seeded with bacteria of the OP50 strain (C to G) or the HT115 strain expressing the double-stranded RNA for vhp-1 (H). The percentages of worms reaching adulthood 4 days after egg laying are shown with standard errors (error bars). WT, wild type.
- FIG. 2.
Effects of the max-2 mutation on KGB-1 and PMK-1 activities. (A) Effects of the max-2 mutation on KGB-1 activity. N2 (wild type [WT]), kgb-1(km21), mek-1(ks54), and max-2(nv162) animals were treated with copper ion (+) or not treated with copper ion (−). Extracts prepared from each animal were immunoblotted with anti-phospho-KGB-1 and anti-KGB-1 antibodies. P-KGB-1, phospho-KGB-1. (B) Effects of max-2 overexpression on KGB-1 activity. N2 wild-type animals and N2 overexpressing either FLAG-MAX-2 (wild type [WT]) or FLAG-MAX-2(K408R) (KR) were treated with copper ion or left alone. Extracts prepared from each animal were immunoblotted with anti-phospho-KGB-1, anti-KGB-1 and anti-FLAG antibodies. (C) Effects of the max-2 mutation on PMK-1 activity. Extracts prepared from each animal were immunoblotted with anti-phospho-p38 MAPK and anti-PMK-1 antibodies. The position of PMK-1 is indicated by an arrow. P-PMK-1, phospho-PMK-1.
- FIG. 3.
MAX-2 interacts with and phosphorylates MLK-1. (A) Interaction of MAX-2 with MLK-1. COS-7 cells were transfected with expression vectors encoding FLAG-MAX-2 (wild type [WT]), FLAG-MAX-2(K408R) (KR), HA-MLK-1 (WT), HA-MLK-1(K193R) (KR), and HA-MLK-1(S355A) (SA) as indicated. Whole-cell extracts (WCE) and immunoprecipitated complexes obtained with anti-FLAG antibodies (IP : FLAG) were analyzed by Western blotting (WB). (B) Comparison of kinase subdomains VII and VIII between C. elegans MLK-1 and human MLK3. Identical residues (black background) and similar residues (gray background) are indicated. The phosphorylated residue of human MLK3 is indicated by an asterisk. (C) Phosphorylation of MLK-1 by MAX-2. COS-7 cells were transfected with expression vectors encoding FLAG-MAX-2 (WT), FLAG-MAX-2(K408R) (KR), HA-MLK-1 (WT), HA-MLK-1(K193R) (KR), and HA-MLK-1(S355A) (SA) as indicated. Whole-cell extracts and immunoprecipitated complexes obtained with anti-HA antibodies were analyzed by Western blotting.
- FIG. 4.
Effects of mutations at the phosphorylation site on MLK-1 activity. (A) Amino acid sequence of kinase subdomains VII and VIII in MEK-1. The phosphorylated residues are indicated by asterisks. (B and C) Phosphorylation of MEK-1 by MLK-1. COS-7 cells were transfected with expression vectors encoding HA-MLK-1 (wild type [WT]), HA-MLK-1(S355E) (SE), HA-MLK-1(S355D) (SD), HA-MLK-1(S355A) (SA), HA-MLK-1(K193R) (KR), FLAG-MEK-1 (WT), FLAG-MEK-1(K99R) (KR), and FLAG-MEK-1(S221A S225A) (SA) as indicated. Whole-cell extracts (WCE) and immunoprecipitated complexes obtained with anti-FLAG antibodies (IP : FLAG) were analyzed by Western blotting (WB).
- FIG. 5.
MLK-1 Ser-355 is essential for heavy metal stress response. Each animal was cultured from embryogenesis on NGM plates containing 100 μM copper ion. The percentages of worms reaching adulthood 4 days after egg laying are shown with standard errors (error bars).
- FIG. 6.
MIG-2 interacts with MAX-2. (A) Comparisons of the CRIB motifs between C. elegans MAX-2 and human PAK1. Identical residues (black background) and similar residues (gray background) are indicated. (B) A two-hybrid assay for the interaction of MAX-2 with MIG-2. The reporter strain L40 was cotransformed with expression vectors encoding LexA DBD-MIG-2(T21N) (GDP), LexA DBD-MIG-2(G16V) (GTP), and GAL4 AD-MAX-2 (MAX-2) as indicated.
- FIG. 7.
Heavy metal stress sensitivity in mig-2 mutants. (A to C) Each animal was cultured from embryogenesis on NGM plates containing 100 μM copper ion. The percentages of worms reaching adulthood 4 days after egg laying are shown with standard errors (error bars).
- FIG. 8.
Effects of the mig-2 mutation on MLK-1 phosphorylation and KGB-1 activity. (A) Phosphorylation of MLK-1 at Ser-355 in animals. N2 (wild-type [WT]), max-2(nv162), and mig-2(mu28) animals integrated with the dpy-7p::ha::mlk-1 transgene were treated with copper ion (+) or not treated with copper ion (−). Extracts prepared from each animal were subjected to immunoprecipitation with anti-HA antibodies (IP:HA). The immunoprecipitates were immunoblotted with anti-phospho-MLK-1 and anti-HA antibodies. In max-2(nv162) mutants, expression of HA-MLK-1 was significantly decreased, presumably by silencing of the transgene (lanes 3 and 4). P-MLK-1, phospho-MLK-1. (B) Effects of the mig-2 mutation on KGB-1 activity. N2 (wild-type), kgb-1(km21), mlk-1(km19), and mig-2(mu28) animals were treated with copper ion or left alone. Extracts prepared from each animal were immunoblotted with anti-phospho-KGB-1 and anti-KGB-1 antibodies. P-KGB-1, phospho-KGB-1.
- FIG. 9.
Proposed model for KGB-1 JNK signaling pathway. The circled P stands for phosphate.
