Figure 1. Mutant MLK-1/SCD-4 MAP3K suppresses dauer constitutive mutations in a manner similar to the mutant SCD-2/ALK Receptor Tyrosine Kinase: A) The gene model of
mlk-1/scd-4. Pink = SH3 domain, green = S/T kinase domain. Scale bar = 1000 bp. The
scd-4(
sa321) mutation causes a G245E change in the kinase domain (also see B, below). The
km19 deletion of the kinase domain (Mizuno, 2004) is indicated by a bracket. B) A subset of an alignment of kinase domains of C. elegans (Ce) MLK-1 harboring the G245E mutation (red), Drosophila (Dm) slpr and human (Hs) MLK3. C) The
mlk-1(
km19) deletion confers a genetic "interaction fingerprint" similar to that of mutations in
scd-2,
hen-1,
soc-1 and
sma-5 and replicates phenotypes conferred by
scd-4(
sa321) (Reiner et al., 2008). Shown is percent dauer formation at 25&#
x2DA;C with the set of reference mutations
daf-8(
e1393),
daf-7(
e1372),
daf-11(
m47) and
daf-2(
e1370) used previously: strong suppression of the Daf-c phenotype conferred by
e1393, moderate suppression of
e1372, weak suppression of
m47 and no suppression of
e1370. Not shown are the Daf-c single mutants assayed in parallel, which were all 100% dauer at 25&#
x2DA;C. P< 0.0001, <0.0001, =0.002 and n.s., respectively (Chi square with Yates correction).
mlk-1(
km19) and N2 wild type yielded no dauers when scored in parallel at 25&#
x2DA;C. D) A schematic of major signaling axes in the dauer regulatory network that are mutated to a dauer constitutive (Daf-c) phenotype. DAF-11/receptor guanylyl cyclase and DAF-7/TGF-beta define major sensory inputs. DAF-8/Smad is lesser player downstream of DAF-7/TGF-beta, and is mutated to a weaker Daf-c phenotype. By genetic interactions, DAF-2/InsR functions downstream of the DAF-7 and DAF-11 axes. E) Pharyngeal GFP expression from the DAF-3/co-Smad-responsive reporter cuIs2 was previously shown to be suppressed by mutation of DAF-7/TGF-beta (Thatcher et al.., 1999; Reiner et al., 2008; left panel) and was restored by additional mutation of SCD-2/ALK (center) and MLK-1/SCD-4/MAP3K (right panel). Scale bar = 10 µm. F) Quantified pixel intensity of pharyngeal GFP signal from cuIs2 in the
daf-7 single mutant and double mutants with
scd-2 and
mlk-1, respectively, measured as arbitrary units on a Nikon eclipse Ni epifluorescent microscope with DS-Fi2 camera (Nikon) and NIS Elements Advanced research, version 4.40 (Nikon). Error bars represent std error and P value was calculated by ANOVA.