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The Csk-binding protein PAG regulates PDGF-induced Src mitogenic signaling via GM1.

Veracini L, Simon V, Richard V, Schraven B, Horejsi V, Roche S, Benistant C - J. Cell Biol. (2008)

Bottom Line: Regulation of SFK mitogenic activity by PAG requires the first N-terminal 97 aa (PAG-N), which include the extracellular and transmembrane domains, palmitoylation sites, and a short cytoplasmic sequence.We also show that PAG-N increases ganglioside GM1 levels at the cell surface and, thus, displaces PDGFR from caveolae, a process that requires the ganglioside-specific sialidase Neu-3.In conclusion, PAG regulates PDGFR membrane partitioning and SFK mitogenic signaling by modulating GM1 levels within caveolae independently from Csk.

View Article: PubMed Central - PubMed

Affiliation: Centre de Recherche en Biochimie Macromoléculare, Centre National de la Recherche Scientifique UMR5237, Universities of Montpellier I and II, 34293 Montpellier, France.

ABSTRACT
Spatial regulation is an important feature of signal specificity elicited by cytoplasmic tyrosine kinases of the Src family (SRC family protein tyrosine kinases [SFK]). Cholesterol-enriched membrane domains, such as caveolae, regulate association of SFK with the platelet-derived growth factor receptor (PDGFR), which is needed for kinase activation and mitogenic signaling. PAG, a ubiquitously expressed member of the transmembrane adaptor protein family, is known to negatively regulate SFK signaling though binding to Csk. We report that PAG modulates PDGFR levels in caveolae and SFK mitogenic signaling through a Csk-independent mechanism. Regulation of SFK mitogenic activity by PAG requires the first N-terminal 97 aa (PAG-N), which include the extracellular and transmembrane domains, palmitoylation sites, and a short cytoplasmic sequence. We also show that PAG-N increases ganglioside GM1 levels at the cell surface and, thus, displaces PDGFR from caveolae, a process that requires the ganglioside-specific sialidase Neu-3. In conclusion, PAG regulates PDGFR membrane partitioning and SFK mitogenic signaling by modulating GM1 levels within caveolae independently from Csk.

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The ganglioside sialidase Neu-3 mediates PAG-N–induced GM1 antimitogenic effects. (A and B) PAG-N–induced GM1 cell surface accumulation requires caveolin and Neu-3 activity. An example (A) of cell surface GM1 level in PAG-N–expressing cells with knocked-down caveolin-1 or Neu-3 is shown. PAG-N–expressing cells were transfected with the indicated siRNA or treated with DANA, as indicated, and were stained with CTxB–Alexa 594. (B) Statistical analysis of the percentage of cells exhibiting specific staining (GM1-positive cells) is shown. (C) Knockdown of Neu-3 mRNA levels. Results are shown as the mean ± SD of three independent experiments. (D) PDGFR immunogold labeling in PAG-N–expressing cells that were treated with 10 μM DANA. A representative example (left) and the statistical analysis (right) are shown. Arrows highlight PDGFR-positive caveolae. (E) PAG-N mitogenic inhibition is reversed by Neu-3 inactivation. NIH 3T3 cells, infected, or not, with the indicated retroviruses, were transfected with the indicated siRNAs (right), serum starved for 30 h with or without 10 μM DANA (left), and then stimulated or not with PDGF in the presence of BrdU. Cells were then fixed and processed for immunofluorescence. The percentage of transfected cells that incorporated BrdU was calculated as described in Material and methods. Results are expressed as the mean ± SD of three to five independent experiments. *, P < 0.05 (using Student's t test).
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fig7: The ganglioside sialidase Neu-3 mediates PAG-N–induced GM1 antimitogenic effects. (A and B) PAG-N–induced GM1 cell surface accumulation requires caveolin and Neu-3 activity. An example (A) of cell surface GM1 level in PAG-N–expressing cells with knocked-down caveolin-1 or Neu-3 is shown. PAG-N–expressing cells were transfected with the indicated siRNA or treated with DANA, as indicated, and were stained with CTxB–Alexa 594. (B) Statistical analysis of the percentage of cells exhibiting specific staining (GM1-positive cells) is shown. (C) Knockdown of Neu-3 mRNA levels. Results are shown as the mean ± SD of three independent experiments. (D) PDGFR immunogold labeling in PAG-N–expressing cells that were treated with 10 μM DANA. A representative example (left) and the statistical analysis (right) are shown. Arrows highlight PDGFR-positive caveolae. (E) PAG-N mitogenic inhibition is reversed by Neu-3 inactivation. NIH 3T3 cells, infected, or not, with the indicated retroviruses, were transfected with the indicated siRNAs (right), serum starved for 30 h with or without 10 μM DANA (left), and then stimulated or not with PDGF in the presence of BrdU. Cells were then fixed and processed for immunofluorescence. The percentage of transfected cells that incorporated BrdU was calculated as described in Material and methods. Results are expressed as the mean ± SD of three to five independent experiments. *, P < 0.05 (using Student's t test).

Mentions: We next sought to try to understand how PAG-N induces cell surface GM1. The effect of PAG-N CC/AA suggested a role for membrane domain localization in this molecular process. Furthermore, we found that caveolin-1 knockdown also reduced the capacity of PAG-N to impact on GM1 (Fig. 7, A and B), suggesting that caveolin or caveolae integrity might be also required. The ganglioside-specific sialidase Neu-3 allows the conversion of di- and trisialidated gangliosides to monosialidated glycolipids such as GM1. Interestingly, this enzyme is localized at the external leaflet of the plasma membrane and interacts with caveolin (Wang et al., 2002; Papini et al., 2004). We thus investigated whether Neu-3 was involved in PAG-N–induced GM1 effects. Neu-3 knockdown strongly reduced GM1 cell surface accumulation elicited by PAG-N (Fig. 7, A–C). We confirmed this result using 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (DANA), which specifically inhibits cell surface–localized Neu-3 (Da Silva et al., 2005). DANA treatment largely reversed both PAG-N–induced GM1 cell surface accumulation and PDGFR displacement from caveolae, confirming Neu-3 knockdown experiments (Fig. 7, A, B, and D). We then addressed the biological impact of Neu-3 activity on PAG-N antimitogenic effect. Neu-3 inhibition, either with DANA (Fig. 7 E, left) or through Neu-3 siRNA transduction (Fig. 7 E, right), largely reversed PAG-N mitogenic inhibition. Collectively, these data point to GM1 as a mediator of PAG-N antimitogenic function. Moreover, these observations stress that Neu-3 controls PAG-N–induced GM1 antimitogenic effects.


The Csk-binding protein PAG regulates PDGF-induced Src mitogenic signaling via GM1.

Veracini L, Simon V, Richard V, Schraven B, Horejsi V, Roche S, Benistant C - J. Cell Biol. (2008)

The ganglioside sialidase Neu-3 mediates PAG-N–induced GM1 antimitogenic effects. (A and B) PAG-N–induced GM1 cell surface accumulation requires caveolin and Neu-3 activity. An example (A) of cell surface GM1 level in PAG-N–expressing cells with knocked-down caveolin-1 or Neu-3 is shown. PAG-N–expressing cells were transfected with the indicated siRNA or treated with DANA, as indicated, and were stained with CTxB–Alexa 594. (B) Statistical analysis of the percentage of cells exhibiting specific staining (GM1-positive cells) is shown. (C) Knockdown of Neu-3 mRNA levels. Results are shown as the mean ± SD of three independent experiments. (D) PDGFR immunogold labeling in PAG-N–expressing cells that were treated with 10 μM DANA. A representative example (left) and the statistical analysis (right) are shown. Arrows highlight PDGFR-positive caveolae. (E) PAG-N mitogenic inhibition is reversed by Neu-3 inactivation. NIH 3T3 cells, infected, or not, with the indicated retroviruses, were transfected with the indicated siRNAs (right), serum starved for 30 h with or without 10 μM DANA (left), and then stimulated or not with PDGF in the presence of BrdU. Cells were then fixed and processed for immunofluorescence. The percentage of transfected cells that incorporated BrdU was calculated as described in Material and methods. Results are expressed as the mean ± SD of three to five independent experiments. *, P < 0.05 (using Student's t test).
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
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fig7: The ganglioside sialidase Neu-3 mediates PAG-N–induced GM1 antimitogenic effects. (A and B) PAG-N–induced GM1 cell surface accumulation requires caveolin and Neu-3 activity. An example (A) of cell surface GM1 level in PAG-N–expressing cells with knocked-down caveolin-1 or Neu-3 is shown. PAG-N–expressing cells were transfected with the indicated siRNA or treated with DANA, as indicated, and were stained with CTxB–Alexa 594. (B) Statistical analysis of the percentage of cells exhibiting specific staining (GM1-positive cells) is shown. (C) Knockdown of Neu-3 mRNA levels. Results are shown as the mean ± SD of three independent experiments. (D) PDGFR immunogold labeling in PAG-N–expressing cells that were treated with 10 μM DANA. A representative example (left) and the statistical analysis (right) are shown. Arrows highlight PDGFR-positive caveolae. (E) PAG-N mitogenic inhibition is reversed by Neu-3 inactivation. NIH 3T3 cells, infected, or not, with the indicated retroviruses, were transfected with the indicated siRNAs (right), serum starved for 30 h with or without 10 μM DANA (left), and then stimulated or not with PDGF in the presence of BrdU. Cells were then fixed and processed for immunofluorescence. The percentage of transfected cells that incorporated BrdU was calculated as described in Material and methods. Results are expressed as the mean ± SD of three to five independent experiments. *, P < 0.05 (using Student's t test).
Mentions: We next sought to try to understand how PAG-N induces cell surface GM1. The effect of PAG-N CC/AA suggested a role for membrane domain localization in this molecular process. Furthermore, we found that caveolin-1 knockdown also reduced the capacity of PAG-N to impact on GM1 (Fig. 7, A and B), suggesting that caveolin or caveolae integrity might be also required. The ganglioside-specific sialidase Neu-3 allows the conversion of di- and trisialidated gangliosides to monosialidated glycolipids such as GM1. Interestingly, this enzyme is localized at the external leaflet of the plasma membrane and interacts with caveolin (Wang et al., 2002; Papini et al., 2004). We thus investigated whether Neu-3 was involved in PAG-N–induced GM1 effects. Neu-3 knockdown strongly reduced GM1 cell surface accumulation elicited by PAG-N (Fig. 7, A–C). We confirmed this result using 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (DANA), which specifically inhibits cell surface–localized Neu-3 (Da Silva et al., 2005). DANA treatment largely reversed both PAG-N–induced GM1 cell surface accumulation and PDGFR displacement from caveolae, confirming Neu-3 knockdown experiments (Fig. 7, A, B, and D). We then addressed the biological impact of Neu-3 activity on PAG-N antimitogenic effect. Neu-3 inhibition, either with DANA (Fig. 7 E, left) or through Neu-3 siRNA transduction (Fig. 7 E, right), largely reversed PAG-N mitogenic inhibition. Collectively, these data point to GM1 as a mediator of PAG-N antimitogenic function. Moreover, these observations stress that Neu-3 controls PAG-N–induced GM1 antimitogenic effects.

Bottom Line: Regulation of SFK mitogenic activity by PAG requires the first N-terminal 97 aa (PAG-N), which include the extracellular and transmembrane domains, palmitoylation sites, and a short cytoplasmic sequence.We also show that PAG-N increases ganglioside GM1 levels at the cell surface and, thus, displaces PDGFR from caveolae, a process that requires the ganglioside-specific sialidase Neu-3.In conclusion, PAG regulates PDGFR membrane partitioning and SFK mitogenic signaling by modulating GM1 levels within caveolae independently from Csk.

View Article: PubMed Central - PubMed

Affiliation: Centre de Recherche en Biochimie Macromoléculare, Centre National de la Recherche Scientifique UMR5237, Universities of Montpellier I and II, 34293 Montpellier, France.

ABSTRACT
Spatial regulation is an important feature of signal specificity elicited by cytoplasmic tyrosine kinases of the Src family (SRC family protein tyrosine kinases [SFK]). Cholesterol-enriched membrane domains, such as caveolae, regulate association of SFK with the platelet-derived growth factor receptor (PDGFR), which is needed for kinase activation and mitogenic signaling. PAG, a ubiquitously expressed member of the transmembrane adaptor protein family, is known to negatively regulate SFK signaling though binding to Csk. We report that PAG modulates PDGFR levels in caveolae and SFK mitogenic signaling through a Csk-independent mechanism. Regulation of SFK mitogenic activity by PAG requires the first N-terminal 97 aa (PAG-N), which include the extracellular and transmembrane domains, palmitoylation sites, and a short cytoplasmic sequence. We also show that PAG-N increases ganglioside GM1 levels at the cell surface and, thus, displaces PDGFR from caveolae, a process that requires the ganglioside-specific sialidase Neu-3. In conclusion, PAG regulates PDGFR membrane partitioning and SFK mitogenic signaling by modulating GM1 levels within caveolae independently from Csk.

Show MeSH
Related in: MedlinePlus