Figure 3.

FBP21 binds with specificity to the splicing factors SmB, SmB′, and U1C. (A) Diagram of SmB GST fusion proteins. The PGM motif that is repeated three times is underlined. The start of GST-SmB (26 aa) and GST-SmB′ (35 aa) fusion proteins is indicated with an arrow. The fusion protein that includes one repeat unit (GST-SmB13) is underlined in bold. (B) Role of the PGM motif in FBP21 interactions. The indicated purified GST fusion proteins were run on SDS/PAGE gels, and one gel was stained with Coomassie blue whereas the other was blotted to nitrocellulose. The symbols GST, U1A, U1C, B′ (SmB′), and B (SmB) refer to the proteins and peptides described in Materials and Methods and above (A). The term GST represents the fusion peptide alone. ld10 is a GST fusion containing a 10-aa proline-rich peptide from formin (11). The filter was probed with radiolabeled GST-FBP21 WW. (C) The specificity of different WW domains for specific proline-rich sequences. The indicated WW domain-containing GST fusion proteins were purified and were run on SDS/PAGE gels. The lanes were loaded with WW domains from FBP11 (11), FBP21 (21), a longer version of FBP21 containing the same WW domains (21L), the distantly related FBP30 (30) protein (12), and the YES-associated protein (YAP) as described in Materials and Methods. One gel was Coomassie blue-stained, and the other three were blotted to nitrocellulose. The filters were probed with radiolabeled GST-SmB′, GST-Ld10, and GST-WBP1 (14), which are representative of PGM, PPLP, and PPPPY proline-rich motifs, respectively.