PACAP type I receptor transactivation is essential for IGF-1 receptor signalling and antiapoptotic activity in neurons

Abstract

Insulin-like growth factor-1 (IGF-1) and pituitary adenylyl cyclase activating polypeptide (PACAP) are both potent neurotrophic and antiapoptotic factors, which exert their effects via phosphorylation cascades initiated by tyrosine kinase and G-protein-coupled receptors, respectively. Here, we have adapted a recently described phosphoproteomic approach to neuronal cultures to characterize the phosphoproteomes generated by these neurotrophic factors. Unexpectedly, IGF-1 and PACAP increased the phosphorylation state of a common set of proteins in neurons. Using PACAP type 1 receptor (PAC1R) null mice, we showed that IGF-1 transactivated PAC1Rs constitutively associated with IGF-1 receptors. This effect was mediated by Src family kinases, which induced PAC1R phosphorylation on tyrosine residues. PAC1R transactivation was responsible for a large fraction of the IGF-1-associated phosphoproteome and played a critical role in the antiapoptotic activity of IGF-1. Hence, in contrast to the general opinion that the trophic activity of IGF-1 is solely mediated by tyrosine kinase receptor-associated signalling, we show that it involves a more complex signalling network dependent on the PAC1 Gs-protein-coupled receptor in neurons.

Figure 1

Principle and validation of the phosphoproteomic approach. (A) Strategy used for the enrichment and identification of phosphoproteins in cortical neurons. Mice cortical neurons grown for 7 days in serum-free medium were exposed for 10 min to either IGF-1 (2.5 ng/ml) or PACAP (1 nM). Neurons were lysed and protein extracts were enriched in PMAC. The phosphoprotein-enriched fraction was then separated onto 2D gels. Proteins were digested by trypsin and identified by MALDI-TOF-MS-based peptide mass fingerprinting (PMF). Protein-derived tryptic phosphopeptides were purified in parallel using TiO₂ microcolumns and analyzed by MALDI-MS/MS to identify phosphorylated residues. (B, C) Efficiency of PMAC to retain phosphoproteins. Total neuronal protein extracts (input), and various fractions (flow through (FT), washing medium (W) and eluate (E)) obtained by PMAC were analyzed by Western blotting using antibodies against phospho-PKA substrates (B), phospho-tyrosine, phospho-Erk1,2 and phospho-Akt and antibodies recognizing Erk1,2 and Akt independently of their phosphorylation state (C). (D) MS analysis of CRMP-4 tryptic digest before (top) and after (bottom) phosphopeptide purification on TiO₂ microcolumns. The identification of the phosphorylated residue in the 1257.570 Da peptide (corresponding to phospho-Ser⁵²² in the CRMP4 sequence) was performed by MALDI-MS/MS.

Figure 2

Phosphorylation of a common set of proteins in cortical neurons exposed to IGF-1 and PACAP. (A) Cortical neurons grown for 7 days in 100-mm culture dishes (∼10⁷ neurons) were incubated for 3 h in phosphate-free HEPES buffer in the absence (top gels) or presence of ³²P-orthophosphoric acid (bottom gels, 5 mCi/dish) before they were exposed or not to either IGF-1 (2.5 ng/ml) or PACAP (1 nM) for 10 min. PMAC phosphoprotein-enriched fractions were separated onto 2D gels and proteins were revealed either by silver staining or autoradiography. Matching between silver-stained and ³²P-labelled spots was performed using the Melanie 5 software (GE Healthcare). Arrows represent phosphoproteins that were identified by PMF. Spot numbers refer to the proteins listed in Supplementary Table S1. Representative gels out of the four gels obtained from different cultures for each experimental condition are illustrated. (B) Pie chart representation of the phosphoprotein categories identified in cortical neurons treated with IGF-1 or PACAP. (C) Schematic representation of the common protein phosphorylation pattern in cortical neurons in response to IGF-1 and PACAP treatments (proteins indexed in A and listed in Supplementary Table S1).

Figure 3

Transactivation of PAC1 receptor by IGF-1 in cortical neurons. (A) PACAP does not transactivate the IGF-1R. Cortical neurons were exposed for 10 min to PACAP (1 nM) or IGF-1 (2.5 ng/ml). IGF-1R activation was assessed by sequential immunoblotting with an antibody against IGF-1Rs phosphorylated on Tyr^1135/1136 and an antibody recognizing the IGF-1R independently of its phosphorylation state. (B) IGF-1-induced PKA activation is mediated by the PAC1R. Cortical neurons from WT or PAC1^−/− mice were exposed for 10 min to IGF-1 in the absence or presence of either Rp-cAMPS (10 μM) or PACAP6-38 (10 nM). PKA activation was assessed by Western blotting using an antibody against phospho-PKA substrates. (C) IGF-1 but not NGF induces cAMP production in a PAC1R-dependent manner. Cortical neurons from WT or PAC1^−/− mice were exposed to IGF-1 or NGF (100 nM) for 5 or 10 min in the presence of 1 mM IBMX and intracellular cAMP content was quantified using an HTRF-based kit. Results, expressed in % of basal cAMP level, are means±s.e.m. of values obtained in five independent experiments. ^**P<0.01, versus corresponding values measured in untreated neurons; ^§§P<0.01, versus corresponding values measured in WT neurons (ANOVA followed by the Student–Newman–Keul test). (D) IGF-1R-associated phosphoproteome in PAC1^−/− neurons. Cortical neurons from PAC1^−/− mice were treated with IGF-1 (2.5 ng/ml) for 10 min and proteins purified by PMAC were separated on 2D gels and stained with silver. The arrowheads indicate the position of spots whose phosphorylation in response to IGF-1 treatment is inhibited in neurons from PAC1^−/− mice. (E) PAC1R transactivation contributes to the phosphorylation of Akt induced by IGF-1 in cortical neurons. Cultured cortical neurons from WT and PAC1^−/− mice were treated for 10 min with IGF-1 (2.5 ng/ml) and the phosphorylation of Akt was analyzed by sequential immunoblotting with antibodies against phospho-Ser⁴⁷³Akt and total Akt. Quantification of Akt phosphorylation was performed by densitometric analysis using the NIH Image software. Data are means±s.e.m. of results obtained in three independent experiments ^*P<0.05 and ^**P<0.01, versus corresponding values measured in untreated neurons; ^§§P<0.01, versus the corresponding value measured in WT neurons (ANOVA followed by the Student–Newman–Keul test).

Figure 4

Role of Src family kinase in IGF-1-mediated transactivation of PAC1 receptors constitutively associated with IGF-1 receptors in cortical neurons. (A) Cortical neurons transfected with the indicated constructs were exposed for 10 min with 2.5 ng/ml IGF-1 in the absence or presence of 1 μM SU6656. Solubilized protein extracts were immunoprecipitated with a polyclonal anti-VSV antibody. Immunoprecipitated proteins were analyzed by immunoblotting using monoclonal antibodies against VSV or phosphotyrosines or the polyclonal IGF-1R antibody. (B) The phosphorylation of Src was analyzed by sequential immunoblotting with antibodies against phospho-Tyr⁴¹⁶ and total Src. Quantification of Src phosphorylation was performed by densitometric analysis using the NIH Image software. Data are means±s.e.m. of results obtained in three independent experiments. ^**P<0.01, versus corresponding values measured in untreated neurons. (C) Neurons were exposed for 10 min in the absence or presence of IGF-1 and kinase inhibitors (also applied for 30 min before the IGF-1 challenge): PP2 (10 μM), PP3 (10 μM), SU6656 (SU, 1 μM), wortmannin (Wort, 100 nM), PD98056 (PD, 10 μM) and GF109203X (GFx, 10 μM). Results, expressed in % of basal cAMP level, are means±s.e.m. of values obtained in five independent experiments. ^**P<0.01, versus corresponding values measured in the absence of IGF-1; ^§§P<0.01, versus IGF-1-induced cAMP production measured in the absence of inhibitor (ANOVA followed by the Student–Newman–Keul test).

Figure 5

Role of PAC1 receptor transactivation in the antiapoptotic activity of IGF-1 in CGNs. (A) CGNs from WT and PAC1^−/− mice, grown for 7 days in a culture medium containing 30 mM KCl+10% fetal calf serum were switched for 8 h to a serum-free medium containing either 30 mM KCl (K30), 5 mM KCl (K5) or K5+IGF-1 (2.5 ng/ml). (B) Cultured CGNs from PAC1^−/− mice, transfected with the pCI/VSV-PAC1R construct, were switched for 8 h to a serum-free medium containing K5+IGF-1. Transfected neurons (arrows) were detected by VSV immunolabelling. Nuclei were stained with Hoechst 33258. Fluorescence photomicrographs of representative fields are illustrated. Dense or fragmented nuclei were considered as apoptotic. Scale bars, 20 μm. (C) The quantification of apoptotic nuclei is shown. Data, expressed as percentage of apoptotic cells, are the means±s.e.m. of results obtained in three independent experiments. ^**P<0.01, versus K5; ^§§P<0.01, versus the corresponding value measured in WT neurons (ANOVA followed by the Student–Newman–Keul test). (D) PAC1R transactivation is involved in the phosphorylation of Akt induced by IGF-1 in CGNs. CGNs from WT and PAC1^−/− mice were switched for 8 h to a serum-free medium containing either K5 or K5+IGF-1. Akt phosphorylation was analyzed by sequential immunoblotting with antibodies against phospho-Ser⁴⁷³Akt and total Akt. Quantification of Akt phosphorylation level was performed by densitometric analysis. Data are means±s.e.m. of values obtained in three independent experiments. ^*P<0.05 and ^**P<0.01, versus corresponding values measured in untreated neurons; ^§§P<0.01, versus the corresponding value measured in WT neurons.