FIGURE 8.

IGF-1R kinase domain models and IGF-1R mutants illustrate potential impact of Ser-1248 phosphorylation. A, IGF-1R kinase domain (PDB code 1K3A) (7) is shown (gray ribbon) with the conformation of its unphosphorylated SFYYS motif indicated (Position-1, green ribbon). The unphosphorylated Ser-1248 and phosphorylated A-loop Tyr-1131 residues are marked (green sphere and orange stick, respectively). The trajectory for connection of the 81-residue C-terminal extension (absent in the crystallographic IGF-1R construct) lies proximal to Tyr(P)-1131. Charge opposition between Ser(P)-1248 and Asp-1091 will drive conformational reorganization of the C-terminal linker region following phosphorylation of Ser-1248 by GSK-3β. The linker may refold around the kinase domain as in position-2 (red ribbon), which is modeled on the corresponding region of the FGFR1 (PDB code 3GQL) (44), with Ser(P)-1248 (red sphere) favorably engaging Lys-1081 and targeting downstream acidic residues (¹²⁵⁹EPEELD¹²⁶⁴) to a basic surface near the binding sites for substrate (blue surface) and nucleotide (stick/orange sphere). Position-2 organization may confer an autoinhibitory function on the C terminus as established for the corresponding region of Tie2 (45, 46). Alternatively, phosphorylation of Ser-1248 may lift the pSFYYS linker region from the surface of the kinase domain as in position-3 (violet ribbon), where Ser(P)-1248 (violet sphere) and C-terminal residues may contribute to protein docking sites. The position-3 conformation is adopted by the C-terminal region of the FGFR1 kinase (PDB code 3GQI), where the cognate residue (Tyr-766) to IGF-1R Ser-1248 is phosphorylated and serves as a binding site for PLCγ Src homology 2 (44). Position-2 and -3 conformations are indicative of feasible structural reorganization arising from Ser-1248 phosphorylation rather than illustrative of definitively established post-phosphorylation conformations for IGF-1R. B, structural detail for the unphosphorylated SFYYS region highlighting Ser-1252 and its proximity to Glu-1254. Charge opposition between these residues following priming phosphorylation on Ser-1252 may destabilize the SFYYS conformation, assisting unfolding and subsequent phosphorylation of Ser-1248 by GSK-3β. C and D, immunoprecipitates of S1248E (C) and K1081G and K1081E mutants (D) mutants transiently expressed in R⁻ cells were incubated with poly(Glu,Tyr) in the presence of [γ-³²P]ATP and assessed for incorporated radioactivity. Data from three independent experiments are presented as the fold change in kinase activity of IGF-1R immunoprecipitates compared with immunoprecipitates from control (R⁻/vector) cells. Error bars reflect the standard deviation, ***, p < 0.001, and **, p < 0.005 were calculated using Student's t test. IGF-1R levels in each assay were determined by immunoblotting.