Hedgehog-stimulated phosphorylation at multiple sites activates Ci by altering Ci–Ci interfaces without full Suppressor of Fused dissociation
Fig 9
Potential trajectories for Ci activation by Fused kinase.
The cartoon illustrates a specific speculative rendition of our general model that full-length Ci-155 is held in an inactive state that masks interaction sites for nuclear transport and co-activator binding through a combination of Ci–Ci and Ci–Su(fu) interactions. The regions of Ci involved in Ci–Ci interactions are proposed to surround functionally important Fu target sites around S218 (pink; 175–230), S286/T294 (light orange; 270–300), and S1230 (dark blue; 1,201–1,271). Speculated interactions are indicated by white dotted lines in A and A’ (A’ and B’ show views of A and B structures from the opposite side). Red wavy lines indicate the known Su(fu) binding sites at residues 230–270 and 1370–1397, together with a third binding site that has been mapped to 832–1020, but is shown as 830–930, preceding the Cos2 CORD binding region (934–1065; dark orange) and CBP binding region (1020–1160; light blue). (A, A’) show potential inert structures if Ci–Ci interactions are intramolecular, stabilizing a structure that allows cooperative binding to Su(fu) (green) at three surfaces. Step 1 shows loss of one Ci–Ci interaction as a consequence of phosphorylation of Fu target sites at S286/T294 and potentially also at another (unidentified or untested) site around the CORD domain to form a structure (B) that remains largely inaccessible to nuclear transport or co-activator proteins. The first step could alternatively disrupt the other speculated Ci–Ci interaction by phosphorylation of S218 and S1230 Fu sites (in all cases, followed by CK1 phosphorylation), again without significant loss of Su(fu) affinity or gain of access to other factors (consistent with the properties of Ci-D1D2). A second step of disrupting the second Ci–Ci interaction through further Ci phosphorylation is hypothesized to disrupt the inert Ci conformation that favors cooperative binding to Su(fu). Here, it is speculated that the Ci conformation (C) still allows significant cooperative binding at the two weaker Su(fu) binding surfaces. The stronger SYGHI binding site (230–270) may then often bind to another Su(fu) molecule, as in (D). Phosphorylation of S1382 and S1385 may then disrupt local Ci–Su(fu) binding in (C) or (D) to form structures (E, F) that no longer favor any cooperative binding of Su(fu) and therefore largely lack associated Su(fu) at the weaker binding surfaces. It is hypothesized that these molecules are no longer protected by Su(fu) from proteolysis, which perhaps depends critically on exposure of C-terminal regions of Ci. (G) shows the potential structure of inert complexes if Ci–Ci interactions are intermolecular, within a dimer. The release of Ci–Ci interactions through Fu phosphorylation to form more accessible structures through steps 1 and 2 could take different paths. Most likely, the Ci–Ci interaction helps a Su(fu) molecule bound to the two weaker sites on one Ci molecule to bind also to the SYGHI region of the second Ci molecule. Loss of these Ci–Ci interactions would then lead to dimer dissociation to form structures shown in (C), possibly followed by binding additional Su(fu) to form (D). Conceivably, however, disrupting Ci–Ci interactions may primarily reduce cooperation between the two weaker Su(fu) binding sites, permitting some formation of a more open dimer structure, as in (H). Further disruption of the interface between Ci 1,370−97 and Su(fu) through additional phosphorylation in step 3 would dissociate this dimeric complex. It is speculated that complexes C, D, or H would have considerable activity and retain substantial protection from proteolysis through Su(fu) binding. Since current evidence suggests that Ci activation per se does not greatly de-stabilize Ci (with reduced ci transcription largely responsible for lower Ci-155 levels in En-expressing cells), it is likely that proceeding to structures (E) and (F) with extensive Su(fu) dissociation is rare. If there is no systematically organized ordering of Fu phosphorylation events, then early phosphorylation of S1382 might potentially lead to greater Ci turnover without effective activation.