Dual function of UNC-51-like kinase 3 (Ulk3) in the Sonic hedgehog signaling pathway

Abstract

The Sonic hedgehog (Shh) signaling pathway controls a variety of developmental processes and is implicated in tissue homeostasis maintenance and neurogenesis in adults. Recently, we identified Ulk3 as an active kinase able to positively regulate Gli proteins, mediators of the Shh signaling in mammals. Here, we provide several lines of evidence that Ulk3 participates in the transduction of the Shh signal also independently of its kinase activity. We demonstrate that Ulk3 through its kinase domain interacts with Suppressor of Fused (Sufu), a protein required for negative regulation of Gli proteins. Sufu blocks Ulk3 autophosphorylation and abolishes its ability to phosphorylate and positively regulate Gli proteins. We show that Shh signaling destabilizes the Sufu-Ulk3 complex and induces the release of Ulk3. We demonstrate that the Sufu-Ulk3 complex, when co-expressed with Gli2, promotes generation of the Gli2 repressor form, and that reduction of the Ulk3 mRNA level in Shh-responsive cells results in higher potency of the cells to transmit the Shh signal. Our data suggests a dual function of Ulk3 in the Shh signal transduction pathway and propose an additional way of regulating Gli proteins by Sufu, through binding to and suppression of Ulk3.

FIGURE 1.

Suppression of Ulk3 gene expression suggests a negative role of Ulk3 in Shh signal transduction. A, constructs expressing Ulk3-specific siRNA1 and siRNA2 were co-expressed with FLAG-tagged Ulk3 and GFP in HEK293 cells and cell lysates were subjected to WB analysis. Both siRNAs suppress expression of Ulk3. B, RCGCs were transiently transfected with the construct expressing Ulk3-specific siRNA1 or siRNA2 and stimulated with SHH protein. The Ulk3 mRNA level was measured by qRT-PCR and normalized with the Hprt mRNA level (left panel) along with measurements of Gli-induced luciferase activity (right panel). The cells transfected with the siRNA-expressing constructs were compared with the cells transfected with empty vector *, p < 0.05; **, p < 0.01 (NE, not expressed). The data are presented as average mean ± S.E. of three measurements obtained from three independent experiments. C, suppression of Ulk3 gene expression is achieved in cell lines stably expressing siRNA1 (clones 1.1, 1.2, and 1.3) and siRNA2 (clones 2.1, 2.2, and 2.3) (left panel). The Ulk3 mRNA level is reduced most effectively (∼50%) in clones 1.1, 1.2, 2.1, and 2.2. The expression level of Ulk3 mRNA, normalized with Hprt mRNA, in the parental cell line Shh-L2 is set as 1 and the values in other cell lines are normalized accordingly. Clones expressing the lower level of Ulk3 mRNA demonstrate a higher induction of Gli-dependent luciferase gene expression under influence of SHH compared with control cell lines Shh-L2, clones 1.4 and 2.4 (right panel). The data are presented as average mean ± S.E. of three independent measurements. D, prolonged propagation of stable cell clone 1.1 expressing the Ulk3-specific siRNA1. Left panel shows Ulk3 mRNA levels and the right panel shows the luciferase activities in cells induced with SHH protein. NA, not analyzed. The data are presented as the average mean ± S.E. of three independent measurements. E, cell lines stably expressing Ulk3-specific siRNA1 and siRNA2 (clones 1.2 and 2.1, respectively) and Shh-L2 cells were transfected at different time points during propagation with Ulk3FLAG and GFP encoding constructs. Cell lysates were analyzed with WB using anti-FLAG and anti-GFP antibodies. The levels of overexpressed proteins were quantified and the Ulk3FLAG protein level was normalized with the level of GFP expression. Ulk3FLAG protein level in Shh-L2 cells at each time point was calculated as 1.

FIGURE 2.

ULK3 physically interacts with SUFU through its KD. A, FLAG-tagged WT and kinase-deficient ULK3 proteins were co-expressed with myc-tagged SUFU in HEK293 cells and immunoprecipitated using M2-a-FLAG or c-myc 9E10 affinity gel, respectively. Immunocomplexes were subjected to WB using a-FLAG and H-300 a-Sufu antibodies. B, schematic presentation of ULK3 proteins tested in the current study. MIT, domain contained within microtubule interacting and trafficking molecules. C, FLAG-tagged WT ULK3 and its deletion mutants were co-expressed with myc-tagged SUFU in HEK293 cells and immunoprecipitated using M2-a-FLAG or c-myc 9E10 affinity gel, respectively. Immunocomplexes were subjected to WB using a-FLAG and H-300 a-Sufu antibodies.

FIGURE 3.

Physical interaction of ULK3 with SUFU abolishes C-terminal autophosphorylation of ULK3. A, upper panel, immunocomplexes obtained using anti-myc antibody and containing myc-tagged SUFU and FLAG-tagged ULK3 or ULK3(K139R) were subjected to in vitro kinase assay in the presence of [γ-³²P]ATP (lanes 1 and 2). Bacterially expressed and purified His-tagged ULK3-Ubi and separately immunopurified FLAG-tagged ULK3 proteins were added to the immunocomplexes prior to the kinase assay (lanes 3–5). Lower panel, the presence of the proteins in the immunoprecipitates was detected by WB using the respective antibody. B, upper panel, FLAG-tagged ULK3 proteins were overexpressed in HEK293 cells, immunopurified using anti-FLAG antibody, and subjected to in vitro kinase assay in the presence of [γ-³²P]ATP. Lower panel shows the presence of the proteins detected by WB using anti-FLAG antibody. C, left panel, FLAG-tagged WT ULK3, kinase-deficient ULK3(K139R), ULK3-CT, and ULK3-KD were overexpressed in HEK293 cells, immunopurified using anti-FLAG antibody, and subjected to in vitro kinase assay in the presence of [γ-³²P]ATP (CT, carboxyl terminus). Right panel, the presence of the proteins in the in vitro kinase reactions was detected by WB using anti-FLAG antibody.

FIGURE 4.

ULK3 relieves the inhibitory effect of SUFU on GLI2 transcriptional activity due to its KD but independently of its kinase activity. A, different amounts of the GLI2GFP-encoding construct and constant amount of the ULK3-encoding construct or respective empty vector were co-overexpressed in Shh-L2 cells and Gli-dependent luciferase activity was measured. Transfected DNA amount was kept constant by compensation of GLI2GFP plasmid with pCI-GFP vector. *, p value < 0.001 and **, p value < 0.05 GLI2 versus GLI2 + ULK3. The data are presented as average mean ± S.E. of three replicates obtained from three independent experiments. B, SUFU and ULK3 (WT, kinase-deficient, or deletion mutants) and GLI2 (250 ng/well) were co-expressed in Shh-L2 cells. Induction of Gli-dependent luciferase activity by empty vector was set as 1. GLI2 induces luciferase activity 46 times above the control (*, p value < 0.001 GLI2 versus empty vector). SUFU represses transcriptional activity of GLI2 (*, p value < 0.001 GLI2 versus GLI2 + SUFU). This repression is partly relieved by WT, kinase-deficient ULK3, and ULK3-KD (*, p value < 0.001, GLI2 + SUFU versus GLI2+SUFU+ULK3/ULK3(K139R)/ULK3-KD) and is not relieved, but more inhibited, by ULK3-CT (**, p value < 0.05, GLI2 + SUFU versus GLI2 + SUFU + ULK3-CT). The data are presented as average mean ± S.E. of three replicates obtained from three independent experiments. C, GLI2 (50 ng/well), ULK3, ULK3-KD, and ULK3-CT or the respective empty vector were co-expressed in Shh-L2 cells. Induction of luciferase activity by the empty vector was set as 1. GLI2 induces luciferase activity 16 times above the vector (*, p value < 0.001). ULK3 and ULK3-KD enhance GLI2 transcriptional activity (*, p value < 0.001). ULK3-KD potentiates the transcriptional activator function of GLI2 stronger than ULK3 (♦, p value < 0.001). Overexpression of ULK3-CT leads to inhibition of GLI2 transcriptional activity (*, p value < 0.001). The data are presented as average mean ± S.E. of three replicates obtained from three independent experiments. D, FLAG-tagged ULK3, ULK3(K139R), ULK3-KD, and GLI2 were overexpressed in HEK293 cells, immunopurified, and subjected to in vitro kinase assay in the presence of [γ-³²P]ATP (left panel). Right panel shows the presence of the proteins detected by WB using anti-FLAG antibody.

FIGURE 5.

ULK3-SUFU complex induces the generation of GLI2^Rep. GLI2GFP fusion protein was expressed alone (lane 2), in combination with WT and kinase-deficient FLAG-tagged ULK3 (lanes 3 and 4), or myc-tagged SUFU (lane 5) in HEK293 cells. GLI2^RepGFP protein is detected in the case of co-expression of GLI2GFP with SUFU combined with ULK3 or ULK3(K139R) by WB using GFP antibody (lanes 6 and 7, respectively).

FIGURE 6.

Interaction of ULK3 with endogenous Sufu is regulated by SHH. FLAG-tagged ULK3 was overexpressed in NIH3T3 cells in the absence or presence of SHH. Endogenous Sufu was immunoprecipitated using C-15 affinity gel. The amount of the precipitated Sufu protein was detected using H-300 a-Sufu antibody. ULK3 protein was detected using a-FLAG antibody. SN, supernatant.

FIGURE 7.

A model of Ulk3 function in Shh signaling pathway. In the absence of Shh, Sufu forms a complex with Ulk3 that possibly interacts with cytoskeleton through its MIT domain. The complex binds full-length Gli2 through Sufu, protecting it from proteosomal degradation. Ulk3 is not catalytically active but plays a regulatory role. The Ulk3-Sufu complex contributes to C-terminal processing of Gli2 probably through recruiting PKA, glycogen synthase kinase 3β (GSK3β), and casein kinase 1 (CK1) kinases to full-length Gli2. This results in generation of Gli2^Rep that may enter the nucleus and inhibit its target gene expression. In the presence of Shh, Ulk3-Sufu-Gli2 complex dissociates, Ulk3 activates itself by autophosphorylation and phosphorylates full-length Gli2. This contributes to generation of Gli2^Act, which translocates to the nucleus and activates its target genes. Other molecule(s) indicated as a question mark (?), for instance, Stk36, may participate in the regulation of Gli2 activity by converting it to transcriptional activator or repressor forms depending on the cellular context and strength of Shh signaling.