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First published online 13 July 2004
doi: 10.1242/jcs.01220

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Phospholipase C-1 is a guanine nucleotide exchange factor for dynamin-1 and enhances dynamin-1-dependent epidermal growth factor receptor endocytosis

¹ Division of Molecular and Life Science, Pohang University of Science and Technology, San 31, Hyojadong, Pohang, Kyungbuk 790-784, Republic of Korea
² Department of Physiology, College of Medicine, Pusan National University, Pusan, 602-739, Republic of Korea
³ Green Cross Institute of Medical Genetics, 164-10 Po Yi-Dong, Seoul, 135-260, Republic of Korea
⁴ Departments of Neuroscience, Pharmacology and Molecular Sciences and Psychiatry, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205 USA

View larger version (30K): [in a new window]   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.

View larger version (44K): [in a new window]   Fig. 2. The SH3 domain of PLC-1 functions as a guanine nucleotide exchange factor (GEF) for dynamin-1 in vivo. (A) PC12 cells stably transfected with PLC-1 constructs under a tet-off system were induced to express wild-type PLC-1. Cells were then treated with 10 ng/ml EGF for the indicated times. Co-immunoprecipitated PLC-1 was analyzed by immunoblotting with anti-PLC-1 antibody. (B) PC12 cells stably transfected with wild-type PLC-1 were metabolically labeled in phosphate-free DMEM with 0.5 mCi/ml [32P]H3PO4 for 4 hours at 37°C and treated with EGF for 5 minutes. Dynamin-1 was immunoprecipitated with anti-dynamin-1 antibody, and bound guanine nucleotides were eluted with 1M KH2PO4 and separated on TLC plates. The ratio of GTP was calculated as GTP/(GTP+GDP) (Jeong et al., 2001; Rosen et al., 1994). (C) PC12 cells transfected with specific siRNA for PLC1 (described in Materials and Methods) were stimulated with EGF for the indicated times. The expression of PLC-ß1, PLC-ß3 and PLC-2 were analyzed by immunoblotting with anti-PLC-ß1, PLC-ß3 and PLC-2 antibodies. (D) PC12 cells were transfected with siRNA for PLC-1 metabolically labeled in phosphate-free DMEM with 0.5 mCi/ml [32P]H3PO4 for 4 hour sat 37°C and treated with EGF for 5 minutes. The bound guanine nucleotides to dynamin-1 were analyzed by TLC. The ratio of GTP was calculated as GTP/(GTP+GDP). Stars indicate a significant difference compared with the control.

View larger version (45K): [in a new window]   Fig. 3. The interaction of PLC-1 with dynamin-1 is essential for its GEF activity for dynamin-1. (A) PC12 cells stably transfected with various PLC-1 constructs were immunoprecipitated with anti-FLAG antibody. The cells were then immunoblotted with anti-dynamin antibody. (B) The stably transfected PC12 cells were metabolically labeled in phosphate-free DMEM with 0.5 mCi/ml [32P]H3PO4 for 4 hours at 37°C and treated with EGF for the indicated times. The bound guanine nucleotides to dynamin-1 were analyzed by TLC. The ratio of GTP was calculated as GTP/(GTP+GDP). (C) PC12 cells overexpressing PLC-1WT were transfected with HA-tagged dynamin-1WT and dynamin-1PRD constructs. Immunoprecipitation with anti-HA antibody was followed by immunoblotting with anti-PLC-1 and anti-HA antibody. (D) PC12 cells overexpressing PLC-1WT were transfected with HA-dynamin-1 and its mutant. The bound guanine nucleotides to dynamin-1 were analyzed by TLC. The ratio of GTP was calculated as GTP/(GTP+GDP). Stars indicate a significant difference compared with the control.

View larger version (53K): [in a new window]   Fig. 4. GEF activity of PLC-1 for dynamin-1 enhances dynamin-1-dependent endocytosis. (A-D) PC12 cells transfected with various PLC-1 constructs (A), siRNA to PLC1 (described in Materials and Methods, #1 and #2) (B), various dynamin-1 constructs (C) or the SH3 domains of PLC-1 and amphiphysin (D) were stimulated with EGF for the indicated times. Cell surface proteins were biotinylated as described in Materials and Methods (Lu et al., 2002). Biotinylated proteins were recovered using streptavidin, and the amount of EGFR recovered was assessed by immunoblotting with anti-EGFR antibody.

View larger version (114K): [in a new window]   Fig. 5. PLC-1's GEF activity potentiates dynamin-1-dependent EGFR endocytosis. PC12 cells transfected with various PLC-1 constructs (A) or PLC-1 siRNA duplexes (B) were incubated with Rhodamine-conjugated EGF at the indicated times and fixed cells were examined by confocal microscopy. Arrowheads indicate the internalized EGF-EGFR vesicles.

View larger version (39K): [in a new window]   Fig. 6. GEF activity of PLC-1 for dynamin-1 upregulates dynamin-dependent ERK activation. PC12 cells transfected with various PLC-1 constructs (A), PLC-1 siRNA duplexes (#1 and #2) (B) or the SH3 domains of PLC-1 and amphiphysin (C) were treated with EGF for indicated times. Activation of ERK was measured by immunoblotting with phospho-ERK1/2 antibody. The total cell lysates were analyzed by immunoblotting with ERK1/2 antibody.

View larger version (19K): [in a new window]   Fig. 7. GEF activity of PLC-1 for dynamin-1 upregulates dynamin-dependent SRE-dependent transcriptional activity. The SRE-luciferase reporter gene was transfected in PC12 cells which were stably transfected with indicated PLC-1 mutant genes (vector, closed squares; PLC-1WT, open squares; PLC-1SH3, open triangles; PLC-1P842L, closed triangles). EGF-induced increase of luciferase activity was quantified as mean ± s.d.

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