SEK-1 MAPKK mediates Ca2+ signaling to determine neuronal asymmetric development in Caenorhabditis elegans

Abstract

The mitogen-activated protein kinase (MAPK) pathway is a highly conserved signaling cascade that converts extracellular signals into various outputs. In Caenorhabditis elegans, asymmetric expression of the candidate odorant receptor STR-2 in either the left or the right of two bilaterally symmetrical olfactory AWC neurons is regulated by axon contact and Ca2+ signaling. We show that the MAPK kinase (MAPKK) SEK-1 is required for asymmetric expression in AWC neurons. Genetic and biochemical analyses reveal that SEK-1 functions in a pathway downstream of UNC-43 and NSY-1, Ca2+/calmodulin-dependent protein kinase II (CaMKII) and MAPK kinase kinase (MAPKKK), respectively. Thus, the NSY-1-SEK-1-MAPK cascade is activated by Ca2+ signaling through CaMKII and establishes asymmetric cell fate decision during neuronal development.

Fig. 1. Caenorhabditis elegans SEK-1 functions as a MAPKK. (A) Suppression of the pbs2Δ mutants by C. elegans SEK-1, JNK-1 and PMK-1. pbs2Δ mutants were transformed with the indicated C. elegans genes. Three independent transformants were streaked onto plates containing 1.2 M sorbitol and incubated at 30°C. The pbs2Δ mutants do not grow in the presence of sorbitol. Model for the yeast osmotic stress-activated Hog1 MAPK pathway is shown in the right panel. (B) Sequence alignment of SEK-1 with mammalian MAPKKs, MKK3, MKK6 and MKK4. Identical amino acids are shaded. The sites of activating phosphorylation in MAPKKs are indicated by asterisks and the kinase subdomains are marked with roman numbers below the sequences. The DDBJ/EMBL/GenBank accession number for the SEK-1 sequence is AB060731. (C) MAPKK activity of SEK-1. HEK 293 cells were transfected with Flag-SEK-1 and Flag-SEK-1(K79R). Anti-Flag antibody immunoprecipitates (IP) were used for in vitro kinase reactions with p38-KI as a substrate (top panel). The immunoprecipitates were immunoblotted (IB) with anti-Flag antibody (second panel). Whole-cell extracts were immunoblotted with anti-phospho-p38 MAPK antibody (third panel) and anti-p38 antibody (bottom panel).

Fig. 2. Effect of the sek-1 mutation on str-2 asymmetry in AWC olfactory neurons. (A) Structure of the sek-1 gene. Exons are indicated by boxes. The shaded and open boxes are the translated and untranslated regions, respectively. The black boxes indicate kinase domains. sek-1(km4) is a 2083-bp deletion mutation in which three exons are missing. (B) The morphology of AWC neurons: a lateral diagram (left panel); a cross-sectional diagram (right panel). (C) Patterns of str-2::gfp expression in wild-type N2 animals, sek-1(km4) mutants, N2 carrying odr3p::nsy-1ΔN (Ex[nsy-1ΔN]) and sek-1(km4) carrying odr-3p::nsy-1ΔN (sek-1 Ex[nsy-1ΔN]). Arrows indicate AWC neurons. Fluorescence posterior to the head is gut auto-fluorescence. The photographs show dorsal views. A, anterior; P, posterior; L, left; R, right.

Fig. 3. SEK-1 MAPKK functions downstream of UNC-43 CaMKII and NSY-1 MAPKKK. (A) Association between SEK-1 and NSY-1. HEK 293 cells were transfected with control vector (–), T7-NSY-1 and Flag-SEK-1(K79R) as indicated. Cell lysates were immunoprecipitated (IP) with anti-Flag antibody. Immunoprecipitates were immunoblotted (IB) with anti-T7 antibody (top panel) and anti-Flag antibody (middle panel). Whole-cell extracts were immunoblotted with anti-T7 antibody (bottom panel). (B) Phosphorylation of SEK-1 by NSY-1. HEK 293 cells were transfected with control vector (–), T7-NSY-1, T7-NSY-1(K703M) and Flag-SEK-1(K79R) as indicated. Immunoprecipitated complexes with anti-Flag antibody were incubated with [γ-³²P]ATP and analyzed by autoradiography (upper panel). The immunoprecipitates were immunoblotted with anti-Flag antibody (lower panel). (C and D) Effects of nsy-1 (C) and unc-43 (D) mutations on SEK-1 activity in C. elegans. The sek-1::gfp (WT) and sek-1(K79R)::gfp (KN) transgenes were injected into wild-type N2 animals, nsy-1(ky400) and unc-43(n1186) mutants as indicated. Cell lysates from each animal were immunoprecipitated with anti-GFP antibody. The immunoprecipitates were used for in vitro kinase reactions with p38-KI (upper panel). The immunoprecipitates were immunoblotted with anti-GFP antibody (lower panel).

Fig. 4. Model for str-2 regulation by the CaMKII–MAPK pathway. See text for details.