Fig. 6. ALK4 phosphorylates TTRAP. (A) SDS-PAGE showing in vitro phosphorylation of TTRAP (arrowhead) and ALK4 autophosphorylation (arrow), when incubated with [-³²P]ATP and ALK4 kinase (+; - denotes no ALK4 added). (B) LC-MS/MS plot depicting in vitro phosphopeptides with T88(phos) and T92(phos). (C,D) Phospho-T88 and phospho-T92 are essential for Ttrap function. (C) mRNA injection, yielding overproduction of TTRAP^T88A/T92A, is compatible with normal gastrulation. Live embryo at 80% epiboly showing normal germ ring (gr) and emerging dorsal axial structures (arrowhead). (D) Injection of TTRAP^T88A/T92A is incapable of rescuing Ttrap^MO defects, as evidenced by thickened germ ring and lack of shield/axial structures. These embryos showed a severe delay in epiboly and appeared as if they had not passed germ ring stage even at 8 hpf (normally 80% epiboly). The defects observed in Ttrap^MO embryos were indistinguishable from Ttrap^MO+TTRAP^T88A/T92A-injected embryos (not shown). Animal views, dorsal to the right.