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Fig. 1. The SH3 domain of PLC-1 functions as a guanine nucleotide exchange factor (GEF) for dynamin-1 in vitro. (A) Purified dynamin-1 (50 nM) together with purified GST (diamonds), GST-PLC-1 SH3 (squares), GST-PLC-1 SH3 (P842L) (circles), GST-Grb2 SH3 (triangles) or GST-amphiphysin SH3 fusion proteins (inverted triangles) (50 nM) were incubated with [³⁵S]GTP-S. At the indicated times, radio-labeled dynamin-1 was measured by a nucleotide exchange assay as described in Materials and Methods. (B) Purified dynamin-1 (50 nM) was preloaded with 1 µM [³H]GDP for 30 minutes at 22°C. Purified GST (diamonds), GST-PLC-1 SH3 (squares), GST-PLC-1 SH3 (P842L) (circles), GST-Grb2 SH3 (triangles) or GST-amphiphysin SH3 fusion proteins (inverted triangles) (50 nM) were added together with 0.5 mM unlabelled GTP at the start of the assay. At the time intervals indicated, the dynamin-1-bound radioactivity was measured by a filter-binding assay. The data is expressed as the percentage of [³H]GDP bound to dynamin-1 before the addition of unlabelled GTP. (C) Purified dynamin-1 (1 µM) and purified GST (triangles), GST-PLC-1 SH3 (squares) or GST-PLC-1 SH3 (P842L) (squares) were incubated with radioactive labeled [³⁵S]GTP-S. At the indicated dose of GST-fused proteins, radiolabeled dynamin-1 was measured by a nucleotide exchange assay. (D) Purified dynamin-1 was incubated with GST, GST-PLC-1 SH3, GST-PLC-1 SH3 (P842L), GST-Grb2 SH3 or GST-amphiphysin SH3 fusion proteins coupled to glutathione-Sepharose beads. Bound proteins were analyzed by immunoblotting with anti-dynamin-1 antibody.

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