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First published online 15 December 2008
doi: 10.1242/dev.025460

Development 136, 241-251 (2009)
Published by The Company of Biologists 2009
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Homeodomain-interacting protein kinases (Hipks) promote Wnt/Wg signaling through stabilization of β-catenin/Arm and stimulation of target gene expression

Wendy Lee1, Sharan Swarup1, Joanna Chen1, Tohru Ishitani2 and Esther M. Verheyen1,*

1 Department of Molecular Biology and Biochemistry, Simon Fraser University, 8888 University Drive, Burnaby, BC, V5A 1S6 Canada.
2 Division of Cell Regulation Systems, Post-Genome Science Center, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.


Figure 1
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  		Fig. 1. Modulation of hipk affects wing development. (A) Wild-type wing. (B,C) Reducing hipk function by generating somatic hipk4 clones (B) or through expression of UAS-hipk-RNAi in the wing with sd-gal4 (C) both led to the loss of the wing margin (arrow). (D,E) Overexpression of hipk in the wing blade with the omb-gal4 driver caused the formation of an additional wing margin (D) and outgrowths from the ventral side of the wing (E). (F) Misexpressing two copies of hipk led to the formation of additional wing tissue outgrowths.

 

Figure 2
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  		Fig. 2. Increasing the levels of Hipk can compensate for the loss of the Wg signaling. (A) Wild-type wing. (B) UAS-hipk/69B-Gal4. (C,D) Misexpression of hipk produced a mild ectopic vein phenotype similar to what is seen with UAS-armS10/+; bs-gal4/+ (C) or nmoDB24/nmoadk(D). (E) The hypomorphic wg allelic combination wg1/wg1-17 caused a wing-to-notum transformation, which was rescued by co-expression of hipk (F). (G) UAS-DFz2N33/+; 69B-Gal4/+ causes a severely notched wings. (H) UAS-DFz2N33/+; 69B/UAS-hipk. Simultaneously misexpressing hipk rescued the loss of wing tissue caused by ectopic expression of DFz3N33. (I) UAS-DFz2N33/+; dpp-Gal4/+. (J) UAS-DFz2N33/+; dpp-Gal4/hipk4. (K) sd>DAxin causes mild notches and loss of posterior margin bristles that are enhanced by loss of one copy of hipk in 32B>Daxin, hipk3/+ (L).

 

Figure 3
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  		Fig. 3. Hipk promotes Wg target gene expression. Antibody staining for Wg targets was performed in w1118, sd>hipk RNAi and omb>hipk discs. (A-C) Dll protein. (D-F) Sens protein. (G-I) Ac protein.

 

Figure 4
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  		Fig. 4. Reduction of hipk results in loss of stabilized Arm protein. The expression of stabilized Arm was examined in wing discs bearing hipk4 mutant clones. (A) Wild-type third instar wing disc. A' is a magnification of A; A'' is a z-section through the disc. (B-D) hipk4 somatic clones were marked by the absence of GFP (green in C,D). Arm protein levels (red in B,D) were reduced in hipk somatic clones (arrowheads in B,D). B'-E' show higher magnification views of discs; B''-E'' show z-sections to reveal the subcellular localization of Arm. In hipk mutant cells, Arm levels were normal in the adherens junctions (arrowheads in B''). (E-E'') Expressing sd>hipk RNAi reduces overall Arm levels.

 

Figure 5
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  		Fig. 5. Hipk is required to stabilize Arm in vivo. The expression of Arm protein was examined in third instar wing discs. (A) Wild type. Small panels beside and above show z-sections through the disc. (B) omb-Gal4/+; UAS-hipk/+ discs show expansion of the Arm domain. (C) omb-gal4/+; UAS-GFP/+ staining shows that omb-gal4 expression domain. (D) omb-gal4/+; UAS-Myc-ArmS2 wing discs were stained with anti-Myc antibody to monitor the stabilization of exogenous wild-type Arm. Myc-ArmS2 is stabilized in response to high Wg activity. (E) This effect was suppressed in hipk mutant discs, omb-gal4/+; UAS-Myc-ArmS2, hipk3/hipk4. Arrow indicates reduced stabilization of ArmS2 compared with D. (F) omb-gal4/+; UAS-Myc-ArmS10/+. Expression of a degradation resistant form of Arm was visualized with an anti-Myc antibody. Myc-ArmS10 was stabilized throughout the omb-Gal4 expression domain. (G) omb-gal4/+; UAS-Myc-ArmS10/+; hipk3/hipk4. Reducing hipk activity did not affect the stabilization of constitutively active Arm.

 

Figure 6
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  		Fig. 6. Hipk proteins can bind to Tcf/Lef1 and Arm/β-catenin. (A) HEK293T cells were co-transfected with HA-Hipk and Myc-Tcf. Cell lysates were immunoprecipitated (IP) with anti-HA, anti-Myc or IgG (control) antibodies and extracts were visualized by western blotting (WB) using anti-HA or anti-Myc antibodies, for Hipk and Tcf, respectively. (B) Myc-Hipk and HA-Arm plasmids were co-transfected into HEK293T cells. Lysates were incubated with anti-HA, anti-Myc or IgG (control) antibodies and immunoprecipitates were detected through WB with anti-HA or anti Myc, for Arm and Hipk, respectively. (C) Mammalian Hipk2 interacts with both β-catenin and Lef1. Flag-Hipk2 and T7-Lef1 or β-catenin were co-transfected into HeLa cells. Cell lysates were immunoprecipitated with indicated antibodies and protein complexes were visualized by immunoblotting with Flag, T7 and β-catenin. Hipk2 bound to T7-Lef1{Delta}N and T7-Lef1{Delta}C, deletion mutants that lack the β-catenin and HMG-binding domains, respectively.

 

Figure 7
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  		Fig. 7. Hipk phosphorylates Arm. (A) HEK293T cell lysates expressing the indicated constructs were immunoprecipitated with appropriate antibodies and the purified proteins were subjected to in vitro kinase assays. Arm is phosphorylated in the presence of Hipk WT (lane 1), but not with Hipk KD (lane 2). Relative levels of protein used in the kinase assay were visualized by immunoblotting (IB) with indicated antibodies. (Lanes 3-11) Indicated truncations of Arm were subjected to kinase assays and loading controls are indicated. (B) Schematic of the Arm truncations used in the study.

 

Figure 8
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  		Fig. 8. Drosophila Hipk/mouse Hipk2 enhance Wg/Wnt responsive transcription in vitro. (A) Topflash assays in HEK293T cells showed promotion of Drosophila Tcf/Arm-dependent transcription by Hipk in a kinase-dependent manner. Topflash values are indicated on the left in black columns. These values were from the average of three independent transfection experiments. Vectors used for each experiment are as indicated in the figure. The negative control Fopflash values are given on the right in white columns. (B) Hipk2 promotes Lef1-mediated transcription in a kinase-dependent manner. Hipk can also stimulate Topflash in HeLa cells. Transcriptional assays were performed with vertebrate homologues in HeLa cells. Indicated values represent the average of two independent transfection experiments. Results are labeled according to those described in A. (C) Topflash assays were performed in Drosophila S2R+ cells in the absence (lanes 1-3) or presence of Wg-conditioned media (lanes 4-6). Both Hipk (lanes 2, 5) and Hipk2 (lanes 3, 6) enhanced Topflash under both conditions.

 

Figure 9
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  		Fig. 9. Hipk enhances the stability of Arm. (A) Cell lysates from discs expressing omb>hipk showed elevated Arm protein when compared with wild type. (B) Lysates from S2R+ cells transfected with Hipk2 show elevated Arm, compared with control. (C) Protein lysates from HeLa cells transfected with Hipk2 and β-catenin show elevated levels of β-catenin compared with control and after transfection with mouse Hipk2 KD. (D) HEK293T cells expressing the indicated constructs were treated with the protein translational inhibitor cycloheximide (CHX). Whole cell lysates were collected over several time points after treatment and analyzed by western blot. Arm levels were visualized by immunoblotting with anti-HA antibody. Co-expression of Hipk WT, and to a lesser degree Hipk KD, enhanced the stability of Arm. β-Tubulin was used as a loading control.

 

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© The Company of Biologists Ltd 2009