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New perspectives in PDGF receptor downregulation: the main role of phosphotyrosine phosphatases

Paola Chiarugi*, Paolo Cirri, Maria L. Taddei, Doriana Talini, Laura Doria, Tania Fiaschi, Francesca Buricchi, Elisa Giannoni, Guido Camici, Giovanni Raugei and Giampietro Ramponi

Department of Biochemical Sciences of the University of Florence, Italy



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  		Fig. 1. PDGF-r tyrosine phosphorylation, internalisation and ubiquitination during a time-course agonist stimulation. 1x106 NIH3T3 cells were serum starved for 24 hours and then stimulated with 30 ng/ml PDGF-BB. PDGF-r was immunoprecipitated from lysates. (A) An anti-phosphotyrosine immunoblot is shown. The blot was stripped and probed again with anti PDGF-r antibody (B). PDGF-r internalization was evaluated by trypsin treatment as reported in the Materials and Methods. In C, an anti-PDGF-r immunoblot is shown and in D the anti-phosphotyrosine stripping and reprobing is reported. In E, the plots of the normalised tyrosine phosphorylation level of total PDGF-r and endosomic PDGF-r are reported (n=4). In F, the immunoprecipitated total PDGF-r was probed with anti-ubiquitin antibodies.

 


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  		Fig. 2. Amount of PDGF-r upon agonist stimulation that is able to be restimulated. 1x106 NIH3T3 cells were serum starved for 24 hours and then stimulated with 30 ng/ml PDGF-BB. PDGF-r was immunoprecipitated from lysates. (A) Anti-PDGF-r immunoblot. (B) Cells were stimulated with PDGF-BB during two time courses for 10 minutes, 45 minutes, 2 hours and 3 hours for the first set, and for 3 hours, before being washed and restimulated again for 10 minutes, 45 minutes, 2 hours and 3 hours for the second set. The antiphosphotyrosine immunoblot and the anti-PDGF-r normalisation are shown.

 


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  		Fig. 3. The effect of PDGF-r downregulation mechanisms on PDGF-r tyrosine phosphorylation. 1x106 NIH3T3 cells were serum starved for 24 hours and then stimulated with 30 ng/ml PDGF-BB. (A) The hypertonic medium or 5 µM MG132 was added 30 minutes before stimulation, whereas 0.1 mM pervanadate was added together with the growth factor. PDGF-R was immunoprecipitated from lysates, and an antiphosphotyrosine immunoblot was performed. (B) Cells were treated as in A, and the endosomic PDGF-r pool was analysed by trypsin treatment as reported in the Materials and Methods. PDGF-r was immunoprecipitated from lysates, and an antiphosphotyrosine immunoblot was performed. The blot was stripped and reprobed with anti-PDGF-r antibodies for normalisation. (C) As a control for MG132 inhibition of proteasome-mediated proteolysis of PDGF-r, an anti-PDGF-r immunoprecipitation of PDGF-stimulated cells untreated or treated with MG132 was performed (right). The analysis of the accumulation of ubiquitinated but not the degradation PDGF-r was performed using an anti-ubiquitin immunoblot. The effect of sucrose treatment on the inhibition of clathrin-mediated endocytosis was checked. The endosomic PDGF-r pool was evaluated by trypsin treatment, as reported in the Materials and Methods.

 


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  		Fig. 4. MAPK and PI3K pathway activation during PDGF-r downregulation. 1x106 NIH3T3 cells were serum starved for 24 hours and then stimulated with 30 ng/ml PDGF-BB. 0.2 M sucrose hypertonic medium (hyp) or 5 µM MG132 was added 30 minutes before stimulation, whereas 0.1 mM pervanadate (van) was added together with the growth factor. (A) 40 µg of the total protein from lysates was used for an anti-phosphoMAPK immunoblot.

Equalisation was confirmed by stripping the blot and reprobing with anti-MAPK antibodies (data not shown). (B) 1x106 NIH3T3 cells were serum starved for 24 hours, then either stimulated with PDGF for 10 minutes directly in the plastic dish or suspended for 30 minutes and then plated or not onto fibronectin-treated dishes for the indicated times. 40 µg of total protein from lysates was used for an anti-phosphoMAPK immunoblot. Equalisation was confirmed by stripping the blot and reprobing with anti-MAPK antibodies (data not shown). (C) 400 µg of total proteins was used in a PI3K assay as reported in the Materials and Methods. The results are representative of at least three repetitions.

 


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  		Fig. 5. Cell cycle analysis in dnLMW-PTP-expressing cells. (A) 2x105 dnLMW-PTP- or mock-transfected NIH3T3 cells were serum starved for 24 hours and then stimulated with 30 ng/ml PDGF-BB for the indicated times. Cells were then lysed and stained with propidium iodide for cytofluorimetric cell cycle analysis with Cell FIT (Becton Dickinson) software. The results are representative of several repetitions (n=4 for each of five independent clones). (B) 1x106 dnLMW-PTP- or mock-transfected NIH3T3 cells were serum starved for 24 hours and then stimulated with 30 ng/ml PDGF-BB. PDGF-R was immunoprecipitated from lysates, and an antiphosphotyrosine immunoblot was performed. The blot was stripped and reprobed with anti-PDGF-r antibodies for normalisation. The results are representative of at least three experiments.

 


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  		Fig. 6. PDGF-r kinase activity during PTPs inhibition. (A,B) 1x106 NIH3T3 cells were serum starved for 24 hours and then stimulated with 30 ng/ml PDGF-BB and with the growth factor together with 0.1 mM pervanadate. PDGF-r was immunoprecipitated from lysates, and an immunokinase assay was performed as reported in the Materials and Methods. PDGF-r autophosphorylation kinase activity is reported in A and PDGF-r kinase activity towards GST-PLC{gamma}1 is reported in B. The ratio between the densitometric analyses of kinase assays and normalization blots (anti PDGF-r or anti-PLC{gamma}1 immunoblots) is shown in both A and B. (C) 1x106 NIH3T3 cells were serum starved for 24 hours and then stimulated with 30 ng/ml PDGF-BB and with the growth factor together with 0.1 mM pervanadate. PDGF-r was immunoprecipitated from lysates and an anti-phospho-Tyr857 immunoblot was performed. Equalisation was checked by stripping the blot and reprobing with anti-PDGF-r antibodies. The results are the means of at least three experiments.

 


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  		Fig. 7. PDGF-r phosphorylation during inhibition of ROS production. (A) The time course of ROS production in PDGF-stimulated NIH3T3 was checked by DCF-DA oxidation using cytofluorimetric analysis as reported in the Materials and Methods. (B,C) 1x106 NIH3T3 cells were serum starved for 24 hours, pretreated or untreated with 1 µg/ml catalase (B) or 50 µM DPI (C) and then stimulated with 30 ng/ml PDGF-BB. PDGF-r was immunoprecipitated from lysates, and an antiphosphotyrosine immunoblot was performed. Equalisation was checked by stripping the blot and reprobing with anti-PDGF-r antibodies. The ratios of the densitometric analyses of anti-phosphotyrosine and anti-PDGF-r signals are reported in B and C. (D,E) PTP assay of lysates from PDGF-stimulated cells. Cells were pretreated or untreated with DPI or catalase for 30 minutes and then stimulated for the indicated times with PDGF-BB. PTP activity on PNPP or on in vitro 32P-autophosphorylated PDGF-r is shown in D and E, respectively. The results are representative of at least three independent experiments.

 


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  		Fig. 8. PDGF-r and PDGF-r phosphorylation persist for many hours. 1x106 NIH3T3 cells were serum starved for 24 hours then stimulated with 30 ng/ml PDGF-BB for the indicated times. PDGF-r was immunoprecipitated from lysates and an antiphosphotyrosine immunoblot was performed (A). The blot was stripped and reprobed with anti-PDGF-r antibodies (B).

 


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  		Fig. 9. PTP inhibition affects long lasting PDGF signaling. 1x106 NIH3T3 cells were serum starved for 24 hours, then stimulated with 30 ng/ml PDGF-BB for the indicated times. (A) PDGF-r was immunoprecipitated from lysates, and an antiphosphotyrosine immunoblot was performed. Equalisation was checked by stripping the blot and reprobing with anti-PDGF-r antibodies. The ratio between the densitometric analyses of anti-phosphotyrosine and anti-PDGF-r signals is shown. (B) Lysates were used for the PI3K assay as reported in the Materials and Methods.

 

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