Figure 4. Warts directly phosphorylates Mud within its C-terminal coiled-coil domain.

(A) The domain architecture of Mud consists primarily of coiled coil (CC) domains. The most C-terminal of these (Mud^CC; blue) contains a single consensus phosphorylation motif for Wts kinase that closely resembles that found in Yki. Immediately following this domain is the minimal Pins-binding domain (Mud^PBD; yellow). (B) Mud^CC, wild-type (MudCC-WT) or a single S1868A mutant (MudCC-S1868A), were incubated in the absence or presence of GST:Wts kinase together with [γ-32P]-ATP. Radioactive phosphate incorporation was assessed by autoradiography using Kodak BioMax-MS radioisotope film. Inset below: Coomassie stain of reaction inputs showing equal levels of Mud protein between phosphorylation conditions. (C) Identical experiments carried out with the NuMA^CC domain and the corresponding T1677A mutant. (D) Coomassie stained gel indicating homogenous purity of the Mud^CC protein purification. (E) Both Mud^CC and NuMA^CC domains elute from a size-exclusion column at volumes consistent with trimers. Predicted elution volumes were calculated from a standard curve performed on the HiLoad Superdex 200 column (GE Healthcare). (F) Schematic of coiled-coil heptad repeat residue interactions. Both S1868 in Mud^CC and T1677 in NuMA^CC are predicted to reside at ‘e’ positions, which participate in ionic interactions in an ideal coiled-coil. See also Figure S2.