The Ste20 kinase misshapen regulates both photoreceptor axon targeting and dorsal closure, acting downstream of distinct signals

Abstract

We have previously shown that the Ste20 kinase encoded by misshapen (msn) functions upstream of the c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase module in Drosophila. msn is required to activate the Drosophila JNK, Basket (Bsk), to promote dorsal closure of the embryo. A mammalian homolog of Msn, Nck interacting kinase, interacts with the SH3 domains of the SH2-SH3 adapter protein Nck. We now show that Msn likewise interacts with Dreadlocks (Dock), the Drosophila homolog of Nck. dock is required for the correct targeting of photoreceptor axons. We have performed a structure-function analysis of Msn in vivo in Drosophila in order to elucidate the mechanism whereby Msn regulates JNK and to determine whether msn, like dock, is required for the correct targeting of photoreceptor axons. We show that Msn requires both a functional kinase and a C-terminal regulatory domain to activate JNK in vivo in Drosophila. A mutation in a PXXP motif on Msn that prevents it from binding to the SH3 domains of Dock does not affect its ability to rescue the dorsal closure defect in msn embryos, suggesting that Dock is not an upstream regulator of msn in dorsal closure. Larvae with only this mutated form of Msn show a marked disruption in photoreceptor axon targeting, implicating an SH3 domain protein in this process; however, an activated form of Msn is not sufficient to rescue the dock mutant phenotype. Mosaic analysis reveals that msn expression is required in photoreceptors in order for their axons to project correctly. The data presented here genetically link msn to two distinct biological events, dorsal closure and photoreceptor axon pathfinding, and thus provide the first evidence that Ste20 kinases of the germinal center kinase family play a role in axonal pathfinding. The ability of Msn to interact with distinct classes of adapter molecules in dorsal closure and photoreceptor axon pathfinding may provide the flexibility that allows it to link to distinct upstream signaling systems.

FIG. 1

Msn binds the SH3 domains of Dock. (A) Various GST fusion proteins (as indicated) were incubated with lysates from 293 cells transfected with myc epitope-tagged Msn. After washing, bound proteins were separated by sodium dodecyl sulfate–8% polyacrylamide gel electrophoresis and visualized by immunoblotting with the anti-myc antibody 9E10. The level of each GST protein was similar as assessed by Coomassie blue staining (data not shown). PLCγ, phospholipase Cγ; ITK, interleukin 2-inducible T-cell kinase. (B) The yeast two-hybrid system was used to identify the PXXP motif in Msn that mediates binding to the SH3 domains of Dock. L40 yeast cells were transfected with LexA-Msn(wt) or LexA-Msn in which two conserved prolines that match potential consensus SH3 binding motifs were mutated to alanine, with Dock expressed as a fusion protein with the activation domain of GAL4. Interaction was determined by selecting for growth on medium lacking histidine in the presence of 5 mM 3-aminotriazole (top). Transfection efficiency was assessed by selecting for growth on media containing histidine (bottom). As a control, LexA-Msn(P656A, P659A) was shown to bind DTRAF1, indicating that LexA-Msn(P656A, P659A) is able to interact with other targets that bind to a different region on Msn. UTL, uracil-tryptophan-leucine; THULL, tryptophan-histidine-uracil-leucine-lysine. (C) Schematic diagram of Msn.

FIG. 2

msn is required for correct targeting of photoreceptor axons. (A to D, F, and G) Photoreceptor axonal projection patterns in third-instar larvae were visualized with MAb 24B10. (A) Wild type. (B) dock^P1. (C) msn¹⁰², 69B-GAL4/msn¹⁰²; UAS-msn(wt)/+. (D) msn¹⁰², 69B-GAL4/msn¹⁰²; UAS-msn(P656A, P659A)/+. (F) msn¹⁰² mutant clone in a Minute background. (G) dock^P1 mutant clone in a Minute background. Wild-type photoreceptors or photoreceptors from msn third-instar larvae rescued with UAS-msn(wt) fan out evenly upon exiting the optic stalk and form a smooth retinotopic array in both the lamina and medulla (A and C). In contrast, msn mutants rescued with UAS-msn(P656A, P659A), which is unable to bind the SH3 domains of Dock, exhibit a severe defect in axonal projections (D). The defect in photoreceptor axonal projections in msn mutants rescued with UAS-msn(P656A, P659A) is more severe than in dock^P1 mutants (B). (E) Immunostaining with antibodies to Elav. Photoreceptor development appears essentially normal except for a defect near the midline of the eye disc. Photoreceptor cell axons from msn mutant clones (F) fail to terminate at a precise depth in the lamina, resulting in an uneven neuropil. However, growth cones from axons that innervate the medulla appear normal. (G) Removal of dock function from the eye by making clones in a Minute background causes the same phenotype as homozygosity for dock^P1 (B).

FIG. 3

Genetic interaction between msn and dock^P1. All panels show third-instar larval eye-brain complexes stained with 24B10. (A) dock^P1. (B) dock^P1; msn¹⁰²/+. msn enhances the dock phenotype. Twenty-two eye-brain complexes homozygous for dock and heterozygous for msn were compared to 14 complexes homozygous for dock, and a consistent enhancement was seen in all samples. (C) msn¹⁰², 69B-GAL4/msn¹⁰², UAS-msn(Δ332–667). (D) dock^P1; elav-GAL4/UAS-msn(Δ332–667). UAS-msn(Δ332–667) rescues both the defect in dorsal closure (Table 1) and the defect in photoreceptor axonal targeting in msn mutants (C). However, expression of UAS-msn(Δ332–667) fails to rescue photoreceptor axonal targeting in dock^P1 mutant third-instar larvae (D).

FIG. 4

JNK activation by Msn(Δ332–667) in 293 cells. Msn(wt) or various Msn mutant constructs (0.1 μg each) were transfected into 293 cells together with 10 ng of a plasmid expressing a fusion protein of ATF2 and the GAL4 DNA binding domain and 5 μg of a plasmid expressing a GAL4 luciferase reporter. Transfection efficiency was assessed by coexpressing 1 μg of a plasmid expressing β-galactosidase. The means from two experiments performed in triplicate are shown. Luciferase activity is expressed in arbitrary units after being standardized to β-galactosidase activity.