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Sprouty4 is an endogenous negative modulator of TrkA signaling and neuronal differentiation induced by NGF.

Alsina FC, Irala D, Fontanet PA, Hita FJ, Ledda F, Paratcha G - PLoS ONE (2012)

Bottom Line: Using real time PCR, we detect a significant increase in the expression of Spry4 mRNA in response to NGF, indicating that Spry4 could modulate intracellular signaling pathways and biological processes induced by NGF and its receptor TrkA.In this work, we demonstrate that overexpression of wild-type Spry4 causes a significant reduction in MAPK and Rac1 activation and neurite outgrowth induced by NGF.Together, these findings establish a new physiological mechanism through which Spry4 regulates neurite outgrowth reducing not only the MAPK pathway but also restricting Rac1 activation in response to NGF.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular and Cellular Neuroscience, Institute of Cellular Biology and Neuroscience Prof Dr E De Robertis-CONICET, School of Medicine, University of Buenos Aires, Buenos Aires, Argentina.

ABSTRACT
The Sprouty (Spry) family of proteins represents endogenous regulators of downstream signaling pathways induced by receptor tyrosine kinases (RTKs). Using real time PCR, we detect a significant increase in the expression of Spry4 mRNA in response to NGF, indicating that Spry4 could modulate intracellular signaling pathways and biological processes induced by NGF and its receptor TrkA. In this work, we demonstrate that overexpression of wild-type Spry4 causes a significant reduction in MAPK and Rac1 activation and neurite outgrowth induced by NGF. At molecular level, our findings indicate that ectopic expression of a mutated form of Spry4 (Y53A), in which a conserved tyrosine residue was replaced, fail to block both TrkA-mediated Erk/MAPK activation and neurite outgrowth induced by NGF, suggesting that an intact tyrosine 53 site is required for the inhibitory effect of Spry4 on NGF signaling. Downregulation of Spry4 using small interference RNA knockdown experiments potentiates PC12 cell differentiation and MAPK activation in response to NGF. Together, these findings establish a new physiological mechanism through which Spry4 regulates neurite outgrowth reducing not only the MAPK pathway but also restricting Rac1 activation in response to NGF.

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Sprouty4 restricts Rac1 activation in response to NGF.A) Rac1 activation (Rac1-GTP) in cell lysates of parental PC12 cells and clones overexpressing Spry4 (PC12-Spry4 cells) stimulated with NGF (50 ng/ml) for 10 min. Rac1 activation was assessed by GST-Pak-CRIB pull-down assay, followed by immunoblot (IB) with anti-Rac1 antibodies. The bottom panels show total Rac1 and Myc-tagged Sprouty4 levels present in cell lysates. B) Histogram shows quantification of Rac1 activation. Results are presented as averages ± SEM from three independent experiments. *p<0.05 (Student's t test). C) Co-immunoprecipitation between endogenous Rac1 and Myc-tagged Sprouty4 transiently transfected in Cos cells. Mock and Myc-tagged Sprouty4 transfected cell lysates were immunoprecipitated (IP) with anti-Myc antibodies. Immunoprecipitated proteins were resolved by SDS-PAGE and the filters immunoblotted with anti-Rac1 antibodies. The bottom panel shows equal level of -tubulin in both sample inputs.
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pone-0032087-g003: Sprouty4 restricts Rac1 activation in response to NGF.A) Rac1 activation (Rac1-GTP) in cell lysates of parental PC12 cells and clones overexpressing Spry4 (PC12-Spry4 cells) stimulated with NGF (50 ng/ml) for 10 min. Rac1 activation was assessed by GST-Pak-CRIB pull-down assay, followed by immunoblot (IB) with anti-Rac1 antibodies. The bottom panels show total Rac1 and Myc-tagged Sprouty4 levels present in cell lysates. B) Histogram shows quantification of Rac1 activation. Results are presented as averages ± SEM from three independent experiments. *p<0.05 (Student's t test). C) Co-immunoprecipitation between endogenous Rac1 and Myc-tagged Sprouty4 transiently transfected in Cos cells. Mock and Myc-tagged Sprouty4 transfected cell lysates were immunoprecipitated (IP) with anti-Myc antibodies. Immunoprecipitated proteins were resolved by SDS-PAGE and the filters immunoblotted with anti-Rac1 antibodies. The bottom panel shows equal level of -tubulin in both sample inputs.

Mentions: Rho-like GTPases, including RhoA, Rac1 and Cdc42 are critical proteins in transducing neurotrophin signals to the actin cytoskeleton [16]. In particular, it has been described that neurite outgrowth induced by NGF/TrkA is mediated by Rac1 activation [17], [18]. Since Spry2 has previously been reported to regulate cell migration of rat intestinal epithelial cells by specifically inhibiting the activation of Rac1, but not Cdc42 GTPases [19], we evaluated whether Spry4 could regulate NGF-induced Rac1 activation in PC12 and PC12-Spry4 cells stimulated with NGF (Fig. 3A). The level of active Rac1 (Rac1-GTP) was measured by using an affinity-precipitation assay with the GST-tagged Rac1-GTP interacting binding domain of Pak. As it has been previously described, Rac1 was activated in PC12 cells after 10 min of NGF stimulation. However, the activity of Rac1 was significantly reduced in PC12-Spry4 cells, indicating that Spry4 negatively regulates Rac1 activation triggered by NGF (Fig. 3A and 3B, and Supplementary Fig. S2). Indeed, the specific co-immunoprecipitation between endogenous Rac1 and Myc-tagged Spry4 reported in Fig. 3C, suggests that these two molecules might directly associate and additionally support its functional interaction.


Sprouty4 is an endogenous negative modulator of TrkA signaling and neuronal differentiation induced by NGF.

Alsina FC, Irala D, Fontanet PA, Hita FJ, Ledda F, Paratcha G - PLoS ONE (2012)

Sprouty4 restricts Rac1 activation in response to NGF.A) Rac1 activation (Rac1-GTP) in cell lysates of parental PC12 cells and clones overexpressing Spry4 (PC12-Spry4 cells) stimulated with NGF (50 ng/ml) for 10 min. Rac1 activation was assessed by GST-Pak-CRIB pull-down assay, followed by immunoblot (IB) with anti-Rac1 antibodies. The bottom panels show total Rac1 and Myc-tagged Sprouty4 levels present in cell lysates. B) Histogram shows quantification of Rac1 activation. Results are presented as averages ± SEM from three independent experiments. *p<0.05 (Student's t test). C) Co-immunoprecipitation between endogenous Rac1 and Myc-tagged Sprouty4 transiently transfected in Cos cells. Mock and Myc-tagged Sprouty4 transfected cell lysates were immunoprecipitated (IP) with anti-Myc antibodies. Immunoprecipitated proteins were resolved by SDS-PAGE and the filters immunoblotted with anti-Rac1 antibodies. The bottom panel shows equal level of -tubulin in both sample inputs.
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Related In: Results  -  Collection

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pone-0032087-g003: Sprouty4 restricts Rac1 activation in response to NGF.A) Rac1 activation (Rac1-GTP) in cell lysates of parental PC12 cells and clones overexpressing Spry4 (PC12-Spry4 cells) stimulated with NGF (50 ng/ml) for 10 min. Rac1 activation was assessed by GST-Pak-CRIB pull-down assay, followed by immunoblot (IB) with anti-Rac1 antibodies. The bottom panels show total Rac1 and Myc-tagged Sprouty4 levels present in cell lysates. B) Histogram shows quantification of Rac1 activation. Results are presented as averages ± SEM from three independent experiments. *p<0.05 (Student's t test). C) Co-immunoprecipitation between endogenous Rac1 and Myc-tagged Sprouty4 transiently transfected in Cos cells. Mock and Myc-tagged Sprouty4 transfected cell lysates were immunoprecipitated (IP) with anti-Myc antibodies. Immunoprecipitated proteins were resolved by SDS-PAGE and the filters immunoblotted with anti-Rac1 antibodies. The bottom panel shows equal level of -tubulin in both sample inputs.
Mentions: Rho-like GTPases, including RhoA, Rac1 and Cdc42 are critical proteins in transducing neurotrophin signals to the actin cytoskeleton [16]. In particular, it has been described that neurite outgrowth induced by NGF/TrkA is mediated by Rac1 activation [17], [18]. Since Spry2 has previously been reported to regulate cell migration of rat intestinal epithelial cells by specifically inhibiting the activation of Rac1, but not Cdc42 GTPases [19], we evaluated whether Spry4 could regulate NGF-induced Rac1 activation in PC12 and PC12-Spry4 cells stimulated with NGF (Fig. 3A). The level of active Rac1 (Rac1-GTP) was measured by using an affinity-precipitation assay with the GST-tagged Rac1-GTP interacting binding domain of Pak. As it has been previously described, Rac1 was activated in PC12 cells after 10 min of NGF stimulation. However, the activity of Rac1 was significantly reduced in PC12-Spry4 cells, indicating that Spry4 negatively regulates Rac1 activation triggered by NGF (Fig. 3A and 3B, and Supplementary Fig. S2). Indeed, the specific co-immunoprecipitation between endogenous Rac1 and Myc-tagged Spry4 reported in Fig. 3C, suggests that these two molecules might directly associate and additionally support its functional interaction.

Bottom Line: Using real time PCR, we detect a significant increase in the expression of Spry4 mRNA in response to NGF, indicating that Spry4 could modulate intracellular signaling pathways and biological processes induced by NGF and its receptor TrkA.In this work, we demonstrate that overexpression of wild-type Spry4 causes a significant reduction in MAPK and Rac1 activation and neurite outgrowth induced by NGF.Together, these findings establish a new physiological mechanism through which Spry4 regulates neurite outgrowth reducing not only the MAPK pathway but also restricting Rac1 activation in response to NGF.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular and Cellular Neuroscience, Institute of Cellular Biology and Neuroscience Prof Dr E De Robertis-CONICET, School of Medicine, University of Buenos Aires, Buenos Aires, Argentina.

ABSTRACT
The Sprouty (Spry) family of proteins represents endogenous regulators of downstream signaling pathways induced by receptor tyrosine kinases (RTKs). Using real time PCR, we detect a significant increase in the expression of Spry4 mRNA in response to NGF, indicating that Spry4 could modulate intracellular signaling pathways and biological processes induced by NGF and its receptor TrkA. In this work, we demonstrate that overexpression of wild-type Spry4 causes a significant reduction in MAPK and Rac1 activation and neurite outgrowth induced by NGF. At molecular level, our findings indicate that ectopic expression of a mutated form of Spry4 (Y53A), in which a conserved tyrosine residue was replaced, fail to block both TrkA-mediated Erk/MAPK activation and neurite outgrowth induced by NGF, suggesting that an intact tyrosine 53 site is required for the inhibitory effect of Spry4 on NGF signaling. Downregulation of Spry4 using small interference RNA knockdown experiments potentiates PC12 cell differentiation and MAPK activation in response to NGF. Together, these findings establish a new physiological mechanism through which Spry4 regulates neurite outgrowth reducing not only the MAPK pathway but also restricting Rac1 activation in response to NGF.

Show MeSH
Related in: MedlinePlus