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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.

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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.
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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.

Mentions: 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.


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 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.
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Related In: Results  -  Collection

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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.
Mentions: 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.

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