FIG 2.

Interaction between SH2B1β and STAT3 depends on STAT3 phosphorylation. (A) Domains of STAT3 and STAT3 mutant constructs. (B) PC-3 cells were transiently transfected with GFP-SH2B1β along with STAT3, STAT3-C-FLAG, HA-STAT3-D, STAT3-K685R, STAT3-Y705F, or STAT3-S727A (designated WT, C, D, K685R, Y705F, and S727A). Lysates were immunoprecipitated using anti-IgG or anti-GFP antibody and resolved with SDS-PAGE followed by immunoblotting using antibodies against SH2B1 and STAT3. Representative blots are shown from four independent experiments. Arrowheads indicate the STAT3 protein. An asterisk indicates the nonspecific bands. (C) The overall complex model contains the SH2 domain of SH2B1β (in cyan; PDB code 2HDX) and STAT3β (in magenta; PDB code 1BG1). A higher-magnification view of the boxed region is shown in panels D and E. (D) STAT3 is shown as a magenta ribbon, and the surface electrostatic potential of SH2B1β is also shown. The first (702) and last (716) residues of the phosphotyrosyl tail segment of STAT3 are numbered. N664 and pY705, which are involved in STAT3-SH2B1β complex formation, are shown as sticks (left). The phosphorylated tyrosine of STAT3 provides a negative charge to interact with SH2B1β (middle). The interaction is likely disrupted in the Y705F mutant due to the loss of the negative charge of the phosphate group (right). (E) The residues involved in SH2B1β-STAT3 interaction are shown (left). SH2B1β and STAT3 are shown as cyan and magenta ribbons, respectively. R555 of SH2B1β is surrounded by polar residues Q643, Q644, and N647 at a distance of 4 to 6 Å (middle). The distance is increased to 7 to 8 Å as R555 is mutated to glutamic acid (right). (F) SH2B1β and STAT3 are shown in cyan and magenta ribbons, respectively. The location of K685 of STAT3 is relatively far away from the interacting interface with 16.4 Å relative to E612, the nearest residue of SH2B1β (left). The distance is not affected after the K685 residue was replaced by arginine (right).