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Gq-coupled purinergic receptors inhibit insulin-like growth factor-I/phosphoinositide 3-kinase pathway-dependent keratinocyte migration.

Taboubi S, Garrouste F, Parat F, Pommier G, Faure E, Monferran S, Kovacic H, Lehmann M - Mol. Biol. Cell (2010)

Bottom Line: Purinergic signaling does not affect the formation of the IGF-I receptor/insulin receptor substrate-I/p85 complex, but blocks the activity of a membrane-targeted active p110alpha mutant, indicating that UTP acts downstream of PI3K membrane recruitment.UTP was also found to efficiently attenuate, within few minutes, the IGF-I-induced PI3K-controlled translocation of the actin-nucleating protein cortactin to the plasma membrane.These findings provide new insight into the signaling cross-talk between receptor tyrosine kinase and Galpha((q/11))-coupled receptors, which mediate opposite effects on p110alpha-PI3K activity and keratinocyte migration.

View Article: PubMed Central - PubMed

Affiliation: INSERM UMR 911, Centre de Recherche en Oncologie Biologique et en Oncopharmacologie, Université Aix-Marseille, Marseille 13005, France.

ABSTRACT
Insulin-like growth factor-I (IGF-I) activation of phosphoinositol 3-kinase (PI3K) is an essential pathway for keratinocyte migration that is required for epidermis wound healing. We have previously reported that activation of Galpha((q/11))-coupled-P2Y(2) purinergic receptors by extracellular nucleotides delays keratinocyte wound closure. Here, we report that activation of P2Y(2) receptors by extracellular UTP inhibits the IGF-I-induced p110alpha-PI3K activation. Using siRNA and pharmacological inhibitors, we demonstrate that the UTP antagonistic effects on PI3K pathway are mediated by Galpha((q/11))-and not G((i/o))-independently of phospholipase Cbeta. Purinergic signaling does not affect the formation of the IGF-I receptor/insulin receptor substrate-I/p85 complex, but blocks the activity of a membrane-targeted active p110alpha mutant, indicating that UTP acts downstream of PI3K membrane recruitment. UTP was also found to efficiently attenuate, within few minutes, the IGF-I-induced PI3K-controlled translocation of the actin-nucleating protein cortactin to the plasma membrane. This supports the UTP ability to alter later migratory events. Indeed, UTP inhibits keratinocyte spreading and migration promoted by either IGF-I or a membrane-targeted active p110alpha mutant, in a Galpha(q/11)-dependent manner both. These findings provide new insight into the signaling cross-talk between receptor tyrosine kinase and Galpha((q/11))-coupled receptors, which mediate opposite effects on p110alpha-PI3K activity and keratinocyte migration.

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UTP inhibits the PI3K signaling pathway downstream of p110α-PI3K membrane recruitment. (A) HaCaT keratinocytes were stimulated for 2- and 5-min with UTP (100 μM), IGF-I (50 ng/ml), or both. Immunoprecipitations were performed on cell lysates with an anti-IGF-I receptor (IGF-IR) antibody (top) or anti-IRS1 antibody (bottom). Immunoprecipitates were analyzed by Western blot using anti-phospho-tyrosine antibody (PY) and anti-p85-PI3K antibody (p85), as indicated. Anti-IRS1 (IRS1) and anti-IGF-IR (IGF-IR) antibodies were used as controls. (B) Myr-p110α*-mER–expressing HaCaT clone and vector-transfected clone were treated with either 4-hydroxytamoxifen (4-OHT) or solvent (Ctrl) and then stimulated with IGF-I (50 ng/ml) (IGF) and/or UTP (100 μM; UTP) for 5 min. Cell lysates were analyzed by Western blot using anti-phospho-Akt (p-Akt) and anti-Akt antibody (Akt) as loading control. Data shown are representative of three independent experiments.
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Figure 3: UTP inhibits the PI3K signaling pathway downstream of p110α-PI3K membrane recruitment. (A) HaCaT keratinocytes were stimulated for 2- and 5-min with UTP (100 μM), IGF-I (50 ng/ml), or both. Immunoprecipitations were performed on cell lysates with an anti-IGF-I receptor (IGF-IR) antibody (top) or anti-IRS1 antibody (bottom). Immunoprecipitates were analyzed by Western blot using anti-phospho-tyrosine antibody (PY) and anti-p85-PI3K antibody (p85), as indicated. Anti-IRS1 (IRS1) and anti-IGF-IR (IGF-IR) antibodies were used as controls. (B) Myr-p110α*-mER–expressing HaCaT clone and vector-transfected clone were treated with either 4-hydroxytamoxifen (4-OHT) or solvent (Ctrl) and then stimulated with IGF-I (50 ng/ml) (IGF) and/or UTP (100 μM; UTP) for 5 min. Cell lysates were analyzed by Western blot using anti-phospho-Akt (p-Akt) and anti-Akt antibody (Akt) as loading control. Data shown are representative of three independent experiments.

Mentions: We next attempted to specify the molecular mechanism whereby purinergic signaling interfered with PI3K pathway. Neither IGF-IR surface expression (measured by flow cytometry; data not shown), nor IGF-IR autophosphorylation on tyrosine residues (Figure 3A, top) were modified by UTP treatment. Furthermore, UTP did not reduce tyrosine phosphorylation of IRS-1, the immediate downstream substrate of IGF-IR, and did not change the capacity of IRS-1 to recruit the p85-PI3K regulatory subunit (Figure 3A, bottom). Collectively, these results indicate that UTP did not alter assembly of the IGF-IR/IRS-1/PI3K complex. To investigate further, we examined the impact of UTP on the signaling function of an inducible active form of the p110α catalytic subunit that is targeted to plasma membrane by myristoylation (Myr-p110α*-mER; Leenders et al., 2004; Pankow et al., 2006). Activation of Myr-p110α*-mER protein was induced by addition of 4-OHT. Figure 3B (top) shows that, in empty vector-transfected cells, 4-OHT had no effect on the basal level of Akt phosphorylation. As expected in these cells, UTP still inhibited IGF-I–induced Akt phosphorylation. In agreement with a previous work (Pankow et al., 2006), we observed that Myr-p110α*-mER activation by 4-OHT–induced Akt phosphorylation. Interestingly, this agonist-independent Akt phosphorylation was strongly inhibited by UTP (Figure 3B, bottom). Together, all these results indicate that inhibition of PI3K signaling pathway by UTP occurred downstream of the PI3K recruitment to the plasma membrane.


Gq-coupled purinergic receptors inhibit insulin-like growth factor-I/phosphoinositide 3-kinase pathway-dependent keratinocyte migration.

Taboubi S, Garrouste F, Parat F, Pommier G, Faure E, Monferran S, Kovacic H, Lehmann M - Mol. Biol. Cell (2010)

UTP inhibits the PI3K signaling pathway downstream of p110α-PI3K membrane recruitment. (A) HaCaT keratinocytes were stimulated for 2- and 5-min with UTP (100 μM), IGF-I (50 ng/ml), or both. Immunoprecipitations were performed on cell lysates with an anti-IGF-I receptor (IGF-IR) antibody (top) or anti-IRS1 antibody (bottom). Immunoprecipitates were analyzed by Western blot using anti-phospho-tyrosine antibody (PY) and anti-p85-PI3K antibody (p85), as indicated. Anti-IRS1 (IRS1) and anti-IGF-IR (IGF-IR) antibodies were used as controls. (B) Myr-p110α*-mER–expressing HaCaT clone and vector-transfected clone were treated with either 4-hydroxytamoxifen (4-OHT) or solvent (Ctrl) and then stimulated with IGF-I (50 ng/ml) (IGF) and/or UTP (100 μM; UTP) for 5 min. Cell lysates were analyzed by Western blot using anti-phospho-Akt (p-Akt) and anti-Akt antibody (Akt) as loading control. Data shown are representative of three independent experiments.
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Related In: Results  -  Collection

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Figure 3: UTP inhibits the PI3K signaling pathway downstream of p110α-PI3K membrane recruitment. (A) HaCaT keratinocytes were stimulated for 2- and 5-min with UTP (100 μM), IGF-I (50 ng/ml), or both. Immunoprecipitations were performed on cell lysates with an anti-IGF-I receptor (IGF-IR) antibody (top) or anti-IRS1 antibody (bottom). Immunoprecipitates were analyzed by Western blot using anti-phospho-tyrosine antibody (PY) and anti-p85-PI3K antibody (p85), as indicated. Anti-IRS1 (IRS1) and anti-IGF-IR (IGF-IR) antibodies were used as controls. (B) Myr-p110α*-mER–expressing HaCaT clone and vector-transfected clone were treated with either 4-hydroxytamoxifen (4-OHT) or solvent (Ctrl) and then stimulated with IGF-I (50 ng/ml) (IGF) and/or UTP (100 μM; UTP) for 5 min. Cell lysates were analyzed by Western blot using anti-phospho-Akt (p-Akt) and anti-Akt antibody (Akt) as loading control. Data shown are representative of three independent experiments.
Mentions: We next attempted to specify the molecular mechanism whereby purinergic signaling interfered with PI3K pathway. Neither IGF-IR surface expression (measured by flow cytometry; data not shown), nor IGF-IR autophosphorylation on tyrosine residues (Figure 3A, top) were modified by UTP treatment. Furthermore, UTP did not reduce tyrosine phosphorylation of IRS-1, the immediate downstream substrate of IGF-IR, and did not change the capacity of IRS-1 to recruit the p85-PI3K regulatory subunit (Figure 3A, bottom). Collectively, these results indicate that UTP did not alter assembly of the IGF-IR/IRS-1/PI3K complex. To investigate further, we examined the impact of UTP on the signaling function of an inducible active form of the p110α catalytic subunit that is targeted to plasma membrane by myristoylation (Myr-p110α*-mER; Leenders et al., 2004; Pankow et al., 2006). Activation of Myr-p110α*-mER protein was induced by addition of 4-OHT. Figure 3B (top) shows that, in empty vector-transfected cells, 4-OHT had no effect on the basal level of Akt phosphorylation. As expected in these cells, UTP still inhibited IGF-I–induced Akt phosphorylation. In agreement with a previous work (Pankow et al., 2006), we observed that Myr-p110α*-mER activation by 4-OHT–induced Akt phosphorylation. Interestingly, this agonist-independent Akt phosphorylation was strongly inhibited by UTP (Figure 3B, bottom). Together, all these results indicate that inhibition of PI3K signaling pathway by UTP occurred downstream of the PI3K recruitment to the plasma membrane.

Bottom Line: Purinergic signaling does not affect the formation of the IGF-I receptor/insulin receptor substrate-I/p85 complex, but blocks the activity of a membrane-targeted active p110alpha mutant, indicating that UTP acts downstream of PI3K membrane recruitment.UTP was also found to efficiently attenuate, within few minutes, the IGF-I-induced PI3K-controlled translocation of the actin-nucleating protein cortactin to the plasma membrane.These findings provide new insight into the signaling cross-talk between receptor tyrosine kinase and Galpha((q/11))-coupled receptors, which mediate opposite effects on p110alpha-PI3K activity and keratinocyte migration.

View Article: PubMed Central - PubMed

Affiliation: INSERM UMR 911, Centre de Recherche en Oncologie Biologique et en Oncopharmacologie, Université Aix-Marseille, Marseille 13005, France.

ABSTRACT
Insulin-like growth factor-I (IGF-I) activation of phosphoinositol 3-kinase (PI3K) is an essential pathway for keratinocyte migration that is required for epidermis wound healing. We have previously reported that activation of Galpha((q/11))-coupled-P2Y(2) purinergic receptors by extracellular nucleotides delays keratinocyte wound closure. Here, we report that activation of P2Y(2) receptors by extracellular UTP inhibits the IGF-I-induced p110alpha-PI3K activation. Using siRNA and pharmacological inhibitors, we demonstrate that the UTP antagonistic effects on PI3K pathway are mediated by Galpha((q/11))-and not G((i/o))-independently of phospholipase Cbeta. Purinergic signaling does not affect the formation of the IGF-I receptor/insulin receptor substrate-I/p85 complex, but blocks the activity of a membrane-targeted active p110alpha mutant, indicating that UTP acts downstream of PI3K membrane recruitment. UTP was also found to efficiently attenuate, within few minutes, the IGF-I-induced PI3K-controlled translocation of the actin-nucleating protein cortactin to the plasma membrane. This supports the UTP ability to alter later migratory events. Indeed, UTP inhibits keratinocyte spreading and migration promoted by either IGF-I or a membrane-targeted active p110alpha mutant, in a Galpha(q/11)-dependent manner both. These findings provide new insight into the signaling cross-talk between receptor tyrosine kinase and Galpha((q/11))-coupled receptors, which mediate opposite effects on p110alpha-PI3K activity and keratinocyte migration.

Show MeSH
Related in: MedlinePlus