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First published online 16 November 2004
doi: 10.1242/jcs.01527

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PKA phosphorylation of Src mediates Rap1 activation in NGF and cAMP signaling in PC12 cells

Yutaro Obara^*, Kirstin Labudda, Tara J. Dillon and Philip J. S. Stork

The Vollum Institute, L474, Oregon Health and Science University, 3181 SW Sam Jackson Park Road, Portland, OR 97239, USA

View larger version (27K): [in a new window]   Fig. 1. Detection of phosphorylation of Src at S17. (A) Limited V8 proteolysis of Flag-Src. A diagram of the published digestion pattern for Src is shown (Collett et al., 1979; Roth et al., 1983). The migrations of the fragments a-d are provided in the gel below. Hek293 cells were transfected with Flag-Src and treated with forskolin for 10 minutes or left untreated. Flag-Src was immunoprecipitated and subjected to limited V8 proteolysis as described in Materials and Methods. SDS-PAGE gel pieces were overlaid with the following amounts of V8 protease per well: 1, 0 µg; 2, 0.005 µg; 3, 0.02 µg; 4, 0.05 µg; 5, 0.1 µg. Phosphorylation of Flag-Src at S17 was visualized by immunoblotting with PKA substrate antibody. The migration of protein standards is shown on the left of the blot (kD). The two smallest detectable fragments represent the 16 kD (d) and 18 kD (c) N-terminal fragments, which are circled in both panels. (B) cAMP and isoproterenol induce phosphorylation of endogenous Src in Hek293 cells. Hek293 cells were treated with forskolin/IBMX (F/I) or Isoproterenol (Iso) for the times indicated. Phosphorylation at S17 (pS17) was detected by western blotting with PSAb following immunoprecipitation of endogenous Src. Total Src is shown as a loading control (lower panels). (C) cAMP-induced phosphorylation of endogenous Src in multiple cell types. CHO, PC12 and AtT-20 cells were treated with forskolin/IBMX (F/I) for the times indicated. Phosphorylation at S17 (pS17) was detected following Src immunoprecipitation by western blotting with PSAb. Total Src was detected as a loading control (lower panels).

View larger version (37K): [in a new window]   Fig. 2. cAMP activates Rap1 and ERKs in a PKA and Src tyrosine kinase-dependent manner. (A) Time course of ERK activation by forskolin/IBMX. PC12 cells were stimulated with forskolin/IBMX (F/I) for the indicated times in the presence or absence of H89 or PP2. Activation of endogenous ERKs was determined by western blot (pERK1/2). Total ERK2 levels are shown as a loading control. (B) Time course of ERK activation by 8-CPT. PC12 cells were stimulated with 8-CPT for the indicated periods in the presence or absence of H89 or PP2. Activation of endogenous ERK1/2 was determined by western blot (pERK1/2) as above. Total ERK2 levels are shown as a loading control. (C) cAMP activation of Rap1 via PKA and Src. PC12 cells were stimulated with forskolin/IBMX (F/I) for 20 minutes in the presence or absence of H89 or PP2, and lysates were analyzed for activation of endogenous Rap1 (Rap1-GTP). Total Rap1 levels are shown as a loading control. (D) cAMP does not activate Ras. PC12 cells were stimulated with forskolin/IBMX (F/I) for 10 minutes and lysates were analyzed for activation of endogenous Ras (Ras-GTP) or ERKs (pERK1/2). Total Ras and ERK2 levels are shown as loading controls. (E) 8-CPT does not activate Ras. PC12 cells were stimulated with 8-CPT (15 minutes) or EGF (5 minutes) and lysates were analyzed for activation of endogenous Ras (Ras-GTP). Total Ras levels are also shown.

View larger version (30K): [in a new window]   Fig. 3. Phosphorylation of endogenous Src by PKA is required for Rap1 and ERK activation in PC12 cells. (A) cAMP induction of Src phosphorylation at S17 and Y416 via PKA by cAMP. PC12 cells were stimulated with forskolin/IBMX (F/I) at various time points in the presence or absence of H89 or left untreated (Un) as indicated. Src was immunoprecipitated and analyzed for phosphorylation at S17 and Y416 by using PSAb (pS17) and phospho-Src (pY416) antibody, respectively. Total Src levels are shown as a loading control. (B) Involvement of S17 phosphorylation in Rap1 activation by cAMP. PC12 cells were cotransfected with Flag-Rap1 and pcDNA3 (vector), Flag-Src wild type, Flag-SrcS17A or Flag-SrcS17D, then stimulated with forskolin/IBMX (F/I) for 20 minutes or left untreated (Un). Lysates were analyzed for activation of Rap1 (Flag-Rap1-GTP). Total Flag-Rap1 and Flag-Src are shown as transfection and loading controls. (C) Involvement of S17 phosphorylation in ERK activation by cAMP. PC12 cells were cotransfected with myc-ERK2 and vector, Flag-Src wild type or Flag-SrcS17A (left panels). Cells were then stimulated with forskolin/IBMX (F/I) for 20 minutes or left untreated (Un) and mycERK2 was immunoprecipitated. Cells were also transfected with vector or Flag-SrcS17D as indicated in the right panels. Activation of mycERK2 was determined by western blot (p-myc-ERK2) and total myc-ERK2 levels are shown as a loading control. Total Flag-Src is shown as a transfection control (lower panels).

View larger version (24K): [in a new window]   Fig. 4. NGF activates Rap1 and ERKs in a PKA and Src family kinase-dependent manner. (A) NGF activation of ERKs at late time points is Src-dependent in PC12 cells. PC12 cells were stimulated with NGF or EGF for the indicated periods in the presence or absence of PP2. Activation of endogenous ERKs (pERK1/2) was determined by western blotting. Total ERK2 levels are shown as loading controls. (B) Time course of Rap1 activation by NGF. PC12 cells were stimulated with NGF or EGF for the indicated times or left untreated (Un) and activation of endogenous Rap1 (Rap1-GTP) (upper panel) or ERK1/2 (pERK1/2, lower panel) was determined by western blot. Total Rap1 levels are shown as a loading control (middle panel). (C) PKA and Src are involved in Rap1 activation by NGF. PC12 cells were stimulated with NGF for 30 minutes in the presence or absence of H89 or PP2, or left untreated (Un). Activation of endogenous Rap1 (Rap1-GTP) was determined by western blot. Total Rap1 levels are shown as a loading control (lower panel). (D) PKA and Src-independent activation of Ras by NGF. PC12 cells were stimulated with NGF or EGF for 5 minutes in the presence or absence of H89 or PP2, or left untreated (Un). Lysates were analyzed for activation of endogenous Ras (Ras-GTP). Total Ras levels are shown as a loading control (lower panel).

View larger version (39K): [in a new window]   Fig. 5. S17 within Src is phosphorylated by NGF in a PKA-dependent manner in PC12 cells. (A) Time course of Src phosphorylation by NGF at S17 and Y416. PC12 cells were stimulated with NGF or EGF for the indicated times, or left untreated (Un). After Src immunoprecipitation, precipitates were analyzed for phosphorylation of Src at S17 and Y416. Total Src levels are shown as a loading control (lower panel). (B) PKA-dependent Src phosphorylation by NGF. PC12 cells were stimulated with NGF for 15 minutes in the presence or absence of H89 and Src phosphorylation was analyzed at S17 and Y416 as described in A. Total Src levels are shown as a loading control (lower panel). (C) Inhibition of NGF induction of Src phosphorylation at S17 by Rp-cAMP. PC12 cells were stimulated with NGF for 15 minutes in the presence or absence of Rp-cAMP, and Src phosphorylation was analyzed at S17 (upper panel) as described in A. Total Src levels are shown as a loading control (lower panel).

View larger version (36K): [in a new window]   Fig. 6. S17 phosphorylation of Src is required for NGF activation of Rap1 and ERKs in PC12 cells. (A) Involvement of S17 phosphorylation in Rap1 activation by NGF. PC12 cells were cotransfected with Flag-Rap1 and pcDNA3 vector (Vec), wild-type Flag-Src (WT), or Flag-SrcS17A (17A) as indicated. Cells were stimulated with NGF for 30 minutes or left untreated (Un), and activation of Rap1 (Flag-Rap1-GTP) was determined by western blot. Total Flag-Rap1 and Flag-Src levels are shown. (B) Involvement of S17 phosphorylation in ERK activation at late time points by NGF in PC12 cells. PC12 cells were transfected with myc-ERK2 and pcDNA3 vector (Vec), wild-type Flag-Src (WT), or Flag-SrcS17A (17A) as indicated. After stimulation with NGF for 30 minutes, activation of myc-ERK2 was determined by western blot (p-myc-ERK2, upper panel). Total myc-ERK2 levels and Flag-Src levels are shown. (C) Lack of involvement of S17 phosphorylation in ERK activation at early time points by NGF. PC12 cells were transfected with myc-ERK2 and pcDNA3 vector (vector), wild-type Flag-Src (WT) or Flag-SrcS17A (17A) as indicated. After stimulation with NGF or EGF for 5 minutes, activation of myc-ERK2 was determined by western blot (p-myc-ERK2, upper panel). Total myc-ERK2 levels and Flag-Src levels are shown. (D) Requirement of TrkA in NGF stimulation of S17 phosphorylation and late ERK activation. PC12-nnr5 cells were serum-starved and stimulated with NGF or forskolin/IBMX (F/I) for the indicated times or left untreated (Un). Phosphorylation of Src at S17 was determined following Src immunoprecipitation by western blotting with PSAb. Lysates were also analyzed for activation of ERK (pERK1/2). Total Src and ERK2 levels are shown as loading controls.

View larger version (64K): [in a new window]   Fig. 7. Src S17 phosphorylation is required for neurite outgrowth induced by cAMP in PC12 cells. (A) ERK mediates neurite outgrowth induced by NGF and dbcAMP, but SFKs are involved only in neurite outgrowth induced by cAMP. PC12 cells were incubated with NGF or dibutyryl cAMP (dbcAMP) for 24 hours in the presence or absence of PD98059 or PP2, and neurite outgrowth was evaluated microscopically. PD98059 blocked neurite outgrowth induced by both NGF and dbcAMP, whereas PP2 only blocked outgrowth induced by dbcAMP. (B) Bar graph of mean percentage±s.e. of cells bearing neurite outgrowths when treated as described in A (n=3-5). (C) Src S17A blocks dbcAMP-induced neurite outgrowth, but not NGF-induced neurite outgrowth. PC12 cells were cotransfected with either pcDNA3 (vector) or Src S17A and with EGFP as a marker for transfected cells. The cells were incubated with NGF or dbcAMP for 24 hours. Neurite outgrowth was observed under fluorescence microscopy to identify transfected cells. (D) Bar graph of mean percentage±s.e. of EGFP-positive cells bearing neurite outgrowths when treated as described in C (n=3-5).