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

Monnier V, Ho KS, Sanial M, Scott MP, Plessis A - BMC Dev. Biol. (2002)

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

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratoire de génétique et évolution, Institut Jacques Monod, 2 Place Jussieu, 75005, Paris, France. monnier@ijm.jussieu.fr

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.

Show MeSH
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.
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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.

Mentions: No interaction was detected between GST-Cos2 and Su(fu) (Figure 1A). The same negative result was obtained using GST-Su(fu) and radiolabelled Cos2 (data not shown).


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

Monnier V, Ho KS, Sanial M, Scott MP, Plessis A - BMC Dev. Biol. (2002)

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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC101406&req=5

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.
Mentions: No interaction was detected between GST-Cos2 and Su(fu) (Figure 1A). The same negative result was obtained using GST-Su(fu) and radiolabelled Cos2 (data not shown).

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

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratoire de génétique et évolution, Institut Jacques Monod, 2 Place Jussieu, 75005, Paris, France. monnier@ijm.jussieu.fr

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.

Show MeSH