Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2000 Nov 15;14(22):2893-905.
doi: 10.1101/gad.843900.

Interactions with Costal2 and suppressor of fused regulate nuclear translocation and activity of cubitus interruptus

G Wang  1 , K Amanai, B Wang, J Jiang
Affiliations

Interactions with Costal2 and suppressor of fused regulate nuclear translocation and activity of cubitus interruptus

G Wang et al. Genes Dev. .

Abstract

The Hedgehog (Hh) family of secreted proteins controls many aspects of growth and patterning in animal development. In Drosophila, Hh acts by preventing the formation of a truncated repressor form of Cubitus interruptus (Ci) and stimulating the transcriptional activity of full-length Ci. Here, we provide evidence that Costal2 (Cos2) and Suppressor of Fused [Su(fu)] inhibit Ci by tethering it in the cytoplasm, whereas Hh induces nuclear translocaltion of Ci through Fused (Fu). We have identified a 125 amino acid domain in the C-terminal part of Ci that mediates response to Cos2 inhibition. We show that Cos2 binds Ci, prevents its nuclear import, and inhibits its activity via this domain. We also provide evidence that Su(fu) regulates Ci through two distinct mechanisms: (1) Su(fu) blocks Ci nuclear import through the N-terminal region of Ci, and (2) it inhibits the activity of Ci through a mechanism independent of Ci nuclear translocation. Finally, we show that Cos2 is required for transducing high levels of Hh signaling activity, and it does so by alleviating the blockage of Ci activity imposed by Su(fu).

PubMed Disclaimer

Figures

Figure 1
Figure 1
Cos2 inhibits the activity of uncleavable forms of Ci. Wing discs shown in this and following figures were derived from late third-instar larvae and are oriented with anterior toward the left and ventral up. Wing discs in A,B,E–H were stained with anti-βgal antibody to visualize ptc-lacZ expression (green). Wing discs in C and D were stained with anti-Ci antibody (2A1) that recognizes full-length Ci (red). The genotypes for the wing discs are as follows: (A,C) wild type; (B,D) MS1096, UAS-Cos2; (E) MS1096, UAS-CiU; (F) MS1096, UAS-CiU, UAS-Cos2; (G) MS1096, UAS-Ci-3P; (H) MS1096, UAS-Ci-3P, UAS-Cos2.
Figure 2
Figure 2
Hh and Cos2 have opposing effects on nuclear translocation of Ci. Wing discs in all panels were treated with LMB followed by immunostaining with anti-Ci antibody 2A1 (red) and anti-Armadillo (Arm) antibody (green). Arm shows membrane and cytoplasmic staining. Insets in A′′–D′′ show high magnification views of the regions indicated by corresponding arrows. (A, A′,A′′) A wild-type wing disc showing Ci (red in A and A′′) and Arm (green in A′ and A′′) expression. A compartment cells near the compartment boundary accumulate high levels of Ci in the nucleus (arrow in A; insets in A′′) whereas A compartment cells away from the compartment boundary retain Ci in the cytoplasm. Note that A compartment cells abutting the compartment boundary show low levels of Ci in the nucleus (arrowhead). (B, B′, B′′) A wing disc ectopically expressing tub > y+ > hh. The flip-out cassette was removed in most cells at early third instar by severe heat shock to induce high levels of FLP activity. A compartment cells ectopically expressing hh accumulate Ci in the nucleus regardless of their positions. (C, C′, C′′) A wing disc bearing slimb mutant clones, which are recognized by strong Ci staining (arrow and arrowhead). slimb mutant cells near the A/P compartment boundary accumulate Ci in the nucleus (arrowhead) whereas slimb mutant cells away from the boundary accumulate Ci in the cytoplasm (arrow; inset in C′′). (D, D′, D′′) A wing disc bearing cos2 mutant clones, which are indicated by strong Ci staining. cos2 mutant cells in A compartment accumulate Ci in the nucleus regardless of their positions (arrow and arrowhead; inset in D′′). Note that cos2 mutant cells abutting the A/P compartment boundary (arrowhead in D) show higher levels of nuclear Ci than wild-type cells in the same position (arrowhead in A).
Figure 3
Figure 3
Regulation of the activity of Ci deletion mutants by Cos2. (A) A summary of UAS-Ci constructs analyzed by in vivo coexpression assay. Filled boxes indicate the Zn-finger DNA-binding domain (from aa 440 to aa 620), and open boxes indicate dCBP bind domain (CBP) or the Gal4 activation domain (AD). Numbers indicate the positions of amino acid residues where Ci deletion mutants end. Two copies of HA tag (not shown) are fused at the N terminus of each construct to allow monitoring the expression level. (B, B′, C, C′) Wing discs expressing MS1096/UAS-CiΔN2 (B) or MS1096/UAS-CiΔNC1 (C) show ectopic ptc-lacZ expression uniformly in the wing-pouch region. However, the ectopic ptc-lacZ expression is suppressed by coexpressing UAS-Cos2 (B′ and C′). (D, D′,E, E′) Wing discs expressing MS1096/UAS-CiGA1 (D) or MS1096/UAS-CiGA2 (E) show ectopic ptc-lacZ expression with higher levels in P compartment cells than in A compartment cells. The ectopic ptc-lacZ expression induced by CiGA1 and CiGA2 persists when UAS-Cos2 is coexpressed (D, E′).
Figure 3
Figure 3
Regulation of the activity of Ci deletion mutants by Cos2. (A) A summary of UAS-Ci constructs analyzed by in vivo coexpression assay. Filled boxes indicate the Zn-finger DNA-binding domain (from aa 440 to aa 620), and open boxes indicate dCBP bind domain (CBP) or the Gal4 activation domain (AD). Numbers indicate the positions of amino acid residues where Ci deletion mutants end. Two copies of HA tag (not shown) are fused at the N terminus of each construct to allow monitoring the expression level. (B, B′, C, C′) Wing discs expressing MS1096/UAS-CiΔN2 (B) or MS1096/UAS-CiΔNC1 (C) show ectopic ptc-lacZ expression uniformly in the wing-pouch region. However, the ectopic ptc-lacZ expression is suppressed by coexpressing UAS-Cos2 (B′ and C′). (D, D′,E, E′) Wing discs expressing MS1096/UAS-CiGA1 (D) or MS1096/UAS-CiGA2 (E) show ectopic ptc-lacZ expression with higher levels in P compartment cells than in A compartment cells. The ectopic ptc-lacZ expression induced by CiGA1 and CiGA2 persists when UAS-Cos2 is coexpressed (D, E′).
Figure 4
Figure 4
Binding of Ci fragments to Cos2. Various Ci fragments were fused to the Gal4 activation domain and the fusion constructs were cotransformed into yeast with a bait construct containing full-length Cos2 fused to the LexA DNA-binding domain. Relative strength of binding was measured by a liquid β-galactosidase assay.
Figure 5
Figure 5
Regulating nuclear translocation of Ci deletion mutants by Cos2. Wing discs carrying clones of cells expressing Ci deletion mutants either alone (A–E) or in conjunction with UAS-Cos2 (A′–E′) were treated with LMB followed by immunostaining with anti-Ci antibody (red) and anti-Flag antibody (green). The Cos2 transgene is tagged by Flag epitope. Ci and Cos2 transgenes were expressed using the actin > CD2 > Gal4 driver line. All Ci deletion mutants accumulate in the nucleus after LMB treatment (A–E). However, CiΔN2, CiΔNC1, CiΔNC2 are retained significantly in the cytoplasm (A′–C′) whereas CiGA1 and CiGA2 remain primarily in the nucleus when the UAS-Cos2 transgene is coexpressed in the same cells (D′–E′).
Figure 6
Figure 6
CORD is sufficient to mediate cytoplasmic retention by Cos2. cl-8 cells were transfected with GFPNLS-CORD expressing construct either alone (A) or in conjunction with HA-Cos2 expressing construct (B–D). GFPNLS-CORD expression is shown in green (A,B,D) and HA-Cos2 in red (C,D). In the absence of HA-Cos2, GFPNLS-CORD accumulates in the nucleus (A). When HA-Cos2 is coexpressed, GFPNLS-CORD is sequestered in the cytoplasm and is colocalized with HA-Cos2 (B–D).
Figure 7
Figure 7
Regulation of Ci nuclear translocation by Su(fu) and Fu. All discs in this figure were treated with LMB followed by immunostaining with anti-Ci antibody, 2A1(red in A–E), and anti-Arm antibody (green in D′,E′). Arrowheads in each panel indicate the A/P compartment boundary. (A) A wing disc expressing MS1096/UAS-Ci. A compartment cells contain cytoplasmic Ci at higher levels than nuclear Ci. In contrast, P compartment cells accumulate higher levels of Ci in the nucleus than in the cytoplasm. (B) A Su(fu) mutant wing disc expressing MS1096/UAS-Ci accumulates Ci largely in the nucleus in both A and P compartment cells. (C) A wing disc expressing MS1096/UAS-CiΔN1. CiΔN1 accumulates primarily in the nucleus in both A and P compartments. (D,D′) A fu mutant wing disc. A compartment cells near the A/P compartment boundary retain significant amounts of Ci in the cytoplasm. (E,E′) A wing disc carrying clones of fu cos2 double mutant cells. fu cos2 double mutant cells are recognized by high levels of Ci staining (arrow). fu cos2 mutant cells near the A/P compartment retain little if any Ci in the cytoplasm.
Figure 8
Figure 8
Cos2 transduces high levels of Hh signaling activity by antagonizing Su(fu). All discs were immunostained with anti-En (green) and/or anti-Ci (red) antibodies. Arrowheads in each panel indicate the A/P compartment boundary. (A) A wild-type wing disc. A compartment cells immediately adjacent to the compartment boundary express En and show low levels of Ci staining (arrow). (B) A wing disc carrying cos2 mutant clones. cos2 mutant cells immediately adjacent to the A/P compartment boundary accumulate high levels of Ci and show diminished levels of En expression (big arrowhead). (C) A clone of cos2 mutant cells derived from the A compartment invade into the P compartment (arrow). The cos2 clone is originated from the A compartment because it expresses Ci but not En. (D) A wing disc expressing MS1096/UAS-hh shows uniform En expression in the wing pouch region of the A compartment. (E) A wing disc expressing MS1096/UAS-hh and carrying cos2 mutant clones. cos2 mutant cells situated in the A compartment accumulate high levels of Ci and show diminished levels of En expression (arrows). (F) A wing disc carrying clones of cos2 Su(fu) double mutant cells and triply stained with anti-En (green), anti-Ci (red) and anti-Myc (purple) antibodies. cos2 Su(fu) double mutant cells are recognized by the lack of Myc expression. cos2 Su(fu) double mutant cells immediately adjacent to the A/P compartment boundary (big arrowhead) or distant from the compartment boundary (arrow) express En and show low levels of Ci. Note that the apparent non-uniform En staining along the dorsoventral axis in D and F is because the mutant wing discs are folded and some of the En expressing cells are out of focus.
Figure 9
Figure 9
Model for regulating Ci nuclear import and activity by complex formation with Cos2, Fu, and Su(fu). (A) A drawing of Ci/Cos2/Fu/Su(fu) tetrameric complex bound to microtubules, with full-length Ci (Ci155) represented by a bar. Several functional motifs of Ci are indicated. The stippled box indicates the Cos2 binding domain (CORD); the hatched box indicates the Su(fu) binding domain; the filled box represents the Zn-finger DNA-binding domain (ZF) ; and the open box depicts the dCBP binding domain (CBP). The short vertical lines mark the positions of PKA phosphorylation sites and the arrow indicates the cleavage site. NLS: nuclear localization signal; NES: nuclear export signal; P: PKA phosphorylation sites. (B) Regulation of Ci processing, nuclear import and transcriptional activity. Cos2 and Su(fu) prevent Ci nuclear translocation by tethering it in the cytoplasm whereas Hh stimulates Ci nuclear import through Fu. Su(fu) also affects the activity of full-length Ci after it enters the nucleus. Cos2 promotes proteolytic processing of Ci in conjunction with PKA, Slimb, and proteasome to generate a C-terminally truncated form of Ci (Ci75), which translocates into the nucleus to repress Hh target gene expression. See text for details.
See this image and copyright information in PMC

References

    1. Akimaru H, Chen Y, Dai P, Hou DX, Nonaka M, Smolik SM, Armstrong S, Goodman RH, Ishii S. Drosophila CBP is a co-activator of cubitus interruptus in hedgehog signalling. Nature. 1997;386:735–738. - PubMed
    1. Alexandre C, Jacinto A, Ingham PW. Transcriptional activation of Hedgehog target genes in Drosophila is mediated directly by the Cubitus interruptus protein, a member of the GLI family of the zinc finger DNA-binding proteins. Genes & Dev. 1996;10:2003–2013. - PubMed
    1. Aza-Blanc P, Ramirez-Weber F, Laget M, Schwartz C, Kornberg T. Proteolysis that is inhibited by Hedgehog targets Cubitus interruptus protein to the nucleus and converts it to a repressor. Cell. 1997;89:1043–1053. - PubMed
    1. Basler K, Struhl G. Compartment boundaries and the control of Drosophila limb pattern by hedgehog protein. Nature. 1994;368:208–214. - PubMed
    1. Brand AH, Perrimon N. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development. 1993;118:401–415. - PubMed

Publication types