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. 2002 Mar 19:2:4.
doi: 10.1186/1471-213x-2-4.

Hedgehog signal transduction proteins: contacts of the Fused kinase and Ci transcription factor with the kinesin-related protein Costal2

Véronique Monnier  1 , Karen S Ho, Matthieu Sanial, Matthew P Scott, Anne Plessis
Affiliations

Hedgehog signal transduction proteins: contacts of the Fused kinase and Ci transcription factor with the kinesin-related protein Costal2

Véronique Monnier et al. BMC Dev Biol. .

Abstract

Background: Hedgehog signaling proteins play important roles in development by controlling growth and patterning in various animals including Drosophila and mammals. Hedgehog signaling triggers changes in responsive cells through a novel transduction mechanism that ultimately controls the transcription of specific target genes via the activity of zinc finger transcription factors of the Cubitus interruptus/GLI family. In flies, key Hedgehog signal transduction components have been identified including the kinesin-related protein Costal2, the serinethreonine kinase Fused, and the PEST-containing protein Suppressor of Fused. These proteins control Cubitus interruptus cleavage, nucleo-cytoplasmic localization and activation. In fly embryos, Costal2, Fused, Suppressor of Fused and Cubitus interruptus are associated in at least one cytoplasmic complex, which interacts with the microtubules in a Hedgehog-dependent manner.

Results: Here we identified and mapped direct interactions between Cos2, Fu, and Ci using an in vitro affinity assay and the yeast two-hybrid system.

Conclusions: Our results provide new insights into the possible mechanism of the cytosolic steps of Hedgehog transduction.

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Figures

Figure 1
Figure 1
Direct specific interaction between Cos2 and Fu. (A). GST pull down assay: In vitro translated, 35S-methionine-labeled Fu, Fu1-305, Fu306-805 or Su(fu) before (Input, Lanes 1, 4, 7 and 10) or after incubation with equal amount of, respectively, GST (used as a control; Lanes 2, 5, 8 and 11) or GST-Cos2 (Lanes 3, 6, 9 and 12). Input equals one third of the amount used in the pull down assay. (B). Yeast two hybrid assay: Left: β-galactosidase assay, Right: leucine assay. Each dot corresponds to a yeast diploid coexpressing, respectively, various regions of Cos2 (as shown below) or Rab3 fused to the DNA binding domain of LexA (DBD LexA) (rows I to V) and respectively Fu306-805, Fu437-805, GGTIIβ fused to the transactivation domain B42 (columns 1 to 3). Both assays were performed using RFY231-pSH18-34 (Rows I to IV) or EGY189-pSH18-34 (Row V). The negative control assays with Rab3 give the same result in both strains (Row I and data not shown). In combination with Fureg, Cos538-751 leads to even higher reporter activation than does Cos538-1201. This could be due to numerous causes as differences in stability of the protein fusion, in their nuclear targeting, and/or affinity for Fureg. Cos2: heptad repeats domain (AA643-990), in black Cos2 motor domain (AA172-357) in grey.
Figure 2
Figure 2
Direct specific interaction between Cos2 and Ci. (A). GST pull-down assay: In vitro translated, 35S-methionine-labeled Ci or a truncated form of Ci (Ci1-430) before (Input, Lanes 1 and 4) or after incubation with equal amount of, respectively, GST (Lanes 2 and 5) or GST-Cos2 fusion (Lanes 3 and 6). Ci: zinc finger domain in grey, activation domain in black, the approximate cleavage position is around AA703. (B). Yeast two hybrid β-galactosidase assay between various regions of Cos-2 or Rab3 in fusion with the DBD lex-A (Rows I to V) and respectively GGTIIβ, Ci, Ci1-346 or Ci340-445 in fusion with B42 (Columns 1 to 4). See also legends to Figure 1B and 2A.
Figure 3
Figure 3
Summary of the interaction domains The interactions between Cos2, Ci, Fu, and Fureg described above and in former work [31] are summarized. The interaction between the region 941–1065 of Ci and Cos2 was previously shown by Wang et al. 2000 [23]. The domains of interactions are indicated in lines above or under each protein structure. No interaction could be detected between Cos2 and Sufu (data not shown). See also legends for Figures 1 and 2. M: Cos2 motor domain, HR: Cos2 heptad repeats, DBD:Ci DNA binding domain; NLS: Ci nuclear localisation signal, AD: Ci activation domain.
Figure 4
Figure 4
Model of Hedgehog signaling In the absence of Hh signal, Cos2 and Su(fu) binding to Ci prevents Ci activation and retain it in the cytoplasm. Most of Ci is available for cleavage in a process which is dependent upon its phosphorylation by the PKA and which involves Cos2 and Slimb. Uncleaved, full-length Ci, is actively exported from the nucleus. Upon Hh reception, Fused is activated and acts on Cos2 and Sufu, alleviating thus their negative effect on Ci. As a result, Ci cleavage is reduced, Ci155 nuclear import overcomes its export and Ci is activated. Ci activation requires Cos2 and Fu to antagonize Su(fu) negative effect. Activated nuclear Ci interact with the CBP to fully activate the transcription of Hh target genes.
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