Protein Kinase A-mediated Gating of Neuregulin-dependent ErbB2-ErbB3 Activation Underlies the Synergistic Action of cAMP on Schwann Cell Proliferation
2008; Elsevier BV; Volume: 283; Issue: 49 Linguagem: Inglês
10.1074/jbc.m802318200
ISSN1083-351X
AutoresPaula V. Monje, Gagani Athauda, Patrick M. Wood,
Tópico(s)Toxin Mechanisms and Immunotoxins
ResumoIn Schwann cells (SCs), cyclic adenosine monophosphate (cAMP) enhances the action of neuregulin, the most potent known mitogen for SCs, by synergistically increasing the activation of two crucial signaling pathways: ERK and Akt. However, the underlying mechanism of cross-talk between neuregulin and cAMP signaling remains mostly undefined. Here, we report that the activation of protein kinase A (PKA), but not that of exchange protein activated by cAMP (EPAC), enhances S-phase entry of SCs by synergistically enhancing the ligand-dependent tyrosine phosphorylation/activation of the neuregulin co-receptor, ErbB2-ErbB3. The role of PKA in neuregulin-ErbB signaling was confirmed using PKA inhibitors, pathway-selective cAMP analogs, and natural ligands stimulating PKA activity in SCs, such as adenosine and epinephrine. Two basic observations defined the synergistic action of PKA as "gating" for neuregulin-ErbB signaling: 1) the activation of PKA was not sufficient to induce S-phase entry or the activation of either ErbB2 or ErbB3; and 2) the presence of neuregulin was strictly required to ignite ErbB activation and thereby ERK and Akt signaling. However, PKA directly phosphorylated ErbB2 on Thr-686, a highly conserved intracellular regulatory site that was required for the PKA-mediated synergistic enhancement of neuregulin-induced ErbB2-ErbB3 activation and proliferation in SCs. The gating action of PKA on neuregulin-induced ErbB2-ErbB3 activation has important biological significance, because it insures signal amplification into the ERK and Akt pathways without compromising either the neuregulin dependence or the high specificity of ErbB signaling pathways. In Schwann cells (SCs), cyclic adenosine monophosphate (cAMP) enhances the action of neuregulin, the most potent known mitogen for SCs, by synergistically increasing the activation of two crucial signaling pathways: ERK and Akt. However, the underlying mechanism of cross-talk between neuregulin and cAMP signaling remains mostly undefined. Here, we report that the activation of protein kinase A (PKA), but not that of exchange protein activated by cAMP (EPAC), enhances S-phase entry of SCs by synergistically enhancing the ligand-dependent tyrosine phosphorylation/activation of the neuregulin co-receptor, ErbB2-ErbB3. The role of PKA in neuregulin-ErbB signaling was confirmed using PKA inhibitors, pathway-selective cAMP analogs, and natural ligands stimulating PKA activity in SCs, such as adenosine and epinephrine. Two basic observations defined the synergistic action of PKA as "gating" for neuregulin-ErbB signaling: 1) the activation of PKA was not sufficient to induce S-phase entry or the activation of either ErbB2 or ErbB3; and 2) the presence of neuregulin was strictly required to ignite ErbB activation and thereby ERK and Akt signaling. However, PKA directly phosphorylated ErbB2 on Thr-686, a highly conserved intracellular regulatory site that was required for the PKA-mediated synergistic enhancement of neuregulin-induced ErbB2-ErbB3 activation and proliferation in SCs. The gating action of PKA on neuregulin-induced ErbB2-ErbB3 activation has important biological significance, because it insures signal amplification into the ERK and Akt pathways without compromising either the neuregulin dependence or the high specificity of ErbB signaling pathways. cAMP is a crucial regulator of many cellular processes, including cell proliferation and differentiation. SCs 2The abbreviations used are: SC, Schwann cell; MEK, mitogen-activated protein kinase/ERK kinase; ERK, extracellular signal-regulated kinase; PI3K, phosphatidylinositol 3-kinase; RTK, receptor tyrosine kinase; PKA, protein kinase A; EPAC, exchange protein activated by cAMP; 6-Bnz-cAMP, N6-benzoyladenosine-3′,5′-cyclic monophosphate; 6-PHE-cAMP, N6-phenyladenosine-3′,5′-cyclic monophosphate; 8-CPT-cAMP, 8-(4-chlorophenylthio)-2′-O-methyladenosine-3′,5′-cyclic monophosphate; db-cAMP, N6-2′-O-dibutyryladenosine-3′,5′-cyclic monophosphate; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; EGF, epidermal growth factor; GST, glutathione S-transferase; PI(3,4)P2, l-α-phosphatidylinositol 3,4-bisphosphate; PLL, poly-l-lysine; PDK, 3′-phosphoinositde-dependent kinase; 6-MB-cAMP, N6-monobutyryladenosine-3′,5′-cAMP; PME-cAMP, 8-(4-methoxyphenylthio)-2′-O-methyladenosine-3′,5′-cyclic monophosphate; 8-Br-cAMP, 8-bromoadenosine-3′,5′-cyclic monophosphorothioate. are unique in their capacity to respond to cAMP, because an accumulation of intracellular cAMP enhances polypeptide growth factor-dependent proliferation (1Raff M.C. Abney E. Brockes J.P. Hornby-Smith A. Cell. 1978; 15: 813-822Abstract Full Text PDF PubMed Scopus (210) Google Scholar). In isolated SCs, cAMP-stimulating agents synergistically increase the potency of neuregulin, platelet-derived growth factor, and fibroblast growth factor as mitogenic signals (2Sobue G. Shuman S. Pleasure D. Brain Res. 1986; 362: 23-32Crossref PubMed Scopus (126) Google Scholar, 3Salzer J.L. Bunge R.P. J. Cell Biol. 1980; 84: 739-752Crossref PubMed Scopus (318) Google Scholar, 4Davis J.B. Stroobant P. J. Cell Biol. 1990; 110: 1353-1360Crossref PubMed Scopus (276) Google Scholar, 5Rahmatullah M. Schroering A. Rothblum K. Stahl R.C. Urban B. Carey D.J. Mol. Cell. Biol. 1998; 18: 6245-6252Crossref PubMed Scopus (97) Google Scholar, 6Kim H.A. Ratner N. Roberts T.M. Stiles C.D. J. Neurosci. 2001; 21: 1110-1116Crossref PubMed Google Scholar). Our previous studies indicated that, in SCs, the synergistic effect of cAMP on S-phase entry relies on the ability of this second messenger to enhance the potency and duration of neuregulin-stimulated MEK-ERK and PI3K-Akt activation, which are both required for cell cycle progression. In the absence of neuregulin, increased intracellular cAMP failed to induce the activation of MEK-ERK or PI3K-Akt (7Monje P.V. Bartlett Bunge M. Wood P.M. Glia. 2006; 53: 649-659Crossref PubMed Scopus (83) Google Scholar). Neuregulins comprise an extensive family of growth factors (8Falls D.L. Exp. Cell Res. 2003; 284: 14-30Crossref PubMed Scopus (856) Google Scholar), which are the specific ligands for ErbB/HER family of receptor tyrosine kinases (RTKs) (9Alroy I. Yarden Y. FEBS Lett. 1997; 410: 83-86Crossref PubMed Scopus (655) Google Scholar, 10Buonanno A. Fischbach G.D. Curr. Opin. Neurobiol. 2001; 11: 287-296Crossref PubMed Scopus (432) Google Scholar). A membrane-bound form of neuregulin is a major component of the axonal mitogen that regulates SC proliferation by axonal contact in peripheral nerves (11Morrissey T.K. Levi A.D. Nuijens A. Sliwkowski M.X. Bunge R.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1431-1435Crossref PubMed Scopus (251) Google Scholar, 12Garratt A.N. Britsch S. Birchmeier C. BioEssays. 2000; 22: 987-996Crossref PubMed Scopus (253) Google Scholar). SCs express ErbB2 and ErbB3 isoforms that signal as a heterodimeric complex-activating multiple pathways, including Ras-Raf-MEK-ERK and PI3K-PDK-Akt (12Garratt A.N. Britsch S. Birchmeier C. BioEssays. 2000; 22: 987-996Crossref PubMed Scopus (253) Google Scholar, 13Vartanian T. Goodearl A. Viehover A. Fischbach G. J. Cell Biol. 1997; 137: 211-220Crossref PubMed Scopus (153) Google Scholar). ErbB2 and ErbB3 complement each other to create an effective signal transducer complex. The extracellular domain of ErbB3 is required for binding to neuregulin, and the tyrosine kinase activity of ErbB2 is required for receptor auto- and cross-phosphorylation, inasmuch ErbB2 lacks a binding domain for neuregulin and ErbB3 lacks a catalytically active intracellular kinase domain (14Citri A. Skaria K.B. Yarden Y. Exp. Cell Res. 2003; 284: 54-65Crossref PubMed Scopus (484) Google Scholar). Upon ligand binding, SH3-containing molecules, such as the adaptor protein c-Shc and the regulatory subunit of PI3K (p85), are recruited to specific phosphorylated tyrosine residues on each activated receptor leading to the activation of Ras-ERK and PI3K-Akt, respectively (9Alroy I. Yarden Y. FEBS Lett. 1997; 410: 83-86Crossref PubMed Scopus (655) Google Scholar). Intracellular cAMP directly activates two main effectors: protein kinase A (PKA) and the newly identified exchange protein activated by cAMP (EPAC), an exchange factor for the small GTPase Rap1 (15de Rooij J. Zwartkruis F.J. Verheijen M.H. Cool R.H. Nijman S.M. Wittinghofer A. Bos J.L. Nature. 1998; 396: 474-477Crossref PubMed Scopus (1625) Google Scholar). Together, PKA and EPAC appear to account for most of the effects of cAMP in mammalian cells (15de Rooij J. Zwartkruis F.J. Verheijen M.H. Cool R.H. Nijman S.M. Wittinghofer A. Bos J.L. Nature. 1998; 396: 474-477Crossref PubMed Scopus (1625) Google Scholar, 16Bos J.L. Nat. Rev. Mol. Cell. Biol. 2003; 4: 733-738Crossref PubMed Scopus (414) Google Scholar, 17Kopperud R. Krakstad C. Selheim F. Doskeland S.O. FEBS Lett. 2003; 546: 121-126Crossref PubMed Scopus (173) Google Scholar). Interestingly, cAMP can regulate the flow of signals through other pathways, a function that is referred to as "gating" by cAMP (18Iyengar R. Science. 1996; 271: 461-463Crossref PubMed Scopus (121) Google Scholar). In particular, cAMP has been shown to regulate the Ras-ERK pathway (19Stork P.J. Schmitt J.M. Trends Cell Biol. 2002; 12: 258-266Abstract Full Text Full Text PDF PubMed Scopus (749) Google Scholar). For example, the activation of PKA by cAMP does not affect the proliferation of NIH3T3 cells, but it inhibits Ras-stimulated ERK activity and Ras-mediated transformation (20Wu J. Dent P. Jelinek T. Wolfman A. Weber M.J. Sturgill T.W. Science. 1993; 262: 1065-1069Crossref PubMed Scopus (824) Google Scholar) by phosphorylating Raf1 and reducing its kinase activity (21Hafner S. Adler H.S. Mischak H. Janosch P. Heidecker G. Wolfman A. Pippig S. Lohse M. Ueffing M. Kolch W. Mol. Cell. Biol. 1994; 14: 6696-6703Crossref PubMed Scopus (292) Google Scholar). As mentioned above, the regulation of neuregulin-induced ERK and Akt signaling by cAMP in SCs can be also considered an example of cAMP-mediated gating; however, the underlying mechanism is unknown. Therefore, the goal of this study was to investigate how signals from neuregulin and cAMP interact to regulate ERK and Akt activation and S-phase progression in SCs. Using a combination of pharmacological inhibitors of PKA and pathway-selective cAMP analogs, we found evidence supporting an involvement of PKA, but not EPAC, in increasing the activation of the ErbB2-ErbB3 co-receptor. PKA activity was sufficient to enhance the neuregulin-induced phosphorylation of specific activating tyrosine residues in both ErbB2 and ErbB3 and thereby enhance both MEK-ERK and PI3K-Akt signaling. PKA activity was not sufficient, however, to replace neuregulin to initiate ErbB2 auto- and trans-phosphorylating activity toward ErbB3 or the activation of downstream MEK or PI3K signaling. Yet, PKA directly phosphorylated ErbB2 on at least one highly conserved PKA phospho-acceptor site, Thr-686, a transmodulatory site with a previously suggested role in enhancing the activation of ligand-activated heterodimerizing ErbB2 subpopulations (22Gulliford T. Ouyang X. Epstein R.J. Cell Signal. 1999; 11: 245-252Crossref PubMed Scopus (13) Google Scholar). In this study, we provide evidence indicating that PKA synergistically enhances neuregulin-dependent ErbB2-ErbB3 activation and DNA synthesis in SCs through a mechanism that requires the direct phosphorylation of ErbB2 on Thr-686. We propose a model of ErbB2-ErbB3 regulation by PKA-mediated gating, which guarantees effective signal amplification into key pathways required for proliferation, without compromising the high specificity of the neuregulin-ErbB interaction or downstream signaling events. Reagents—Cell-permeable cAMP analogs (Biolog) were as follows: N6-benzoyladenosine-3′,5′-cyclic monophosphate (6-Bnz-cAMP), N6-phenyladenosine-3′,5′-cyclic monophosphate (6-PHE-cAMP), N6-monobutyryladenosine-3′,5′-cAMP, (6-MB-cAMP), 8-(4-chlorophenylthio)-2′-O-methyladenosine-3′,5′-cyclic monophosphate (8-CPT-cAMP), 8-(4-methoxyphenylthio)-2′-O-methyladenosine-3′,5′-cyclic monophosphate (PME-cAMP), N6-2′-O-dibutyryladenosine-3′,5′-cyclic monophosphate (db-cAMP), 8-bromoadenosine-3′,5′-cyclic monophosphorothioate (8-Br-cAMP), and adenosine-3′,5′-cyclic monophosphorothioate, Rp-isomer. Recombinant heregulin-β1177–244 active peptide was from Genentech (South San Francisco, CA). Recombinant purified platelet-derived growth factor-BB and fibroblast growth factor-1 were from R&D Systems. Forskolin, adenosine, epinephrine, isoproterenol, and H-89 were from Sigma. Cell-permeable myristoylated protein kinase inhibitor peptide 14–22 (Myr-PKI) and l-α-phosphatidyl-inositol-3,4-bisphosphate (PI(3,4)P2) were from Biomol (Plymouth Meeting, PA). ErbB3 antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Total and Phospho-Akt (Ser-473 and Thr-308), -GSK3β (Ser-9), -MEK, -ERK, -ErbB2 (Tyr-1221/22, Tyr-877, and Tyr-1248) polyclonal antibodies, phospho-ErbB3 (Tyr-1289), total phosphotyrosine and phospho-PKA substrate antibodies were from Cell Signaling Technologies (Beverly, MA). P-ErbB2 (Thr-686) was from GeneTex (San Antonio, TX). [3H]thymidine (6.7 Ci/mmol) and Solvable™ were from PerkinElmer Life Sciences (Boston, MA). The expression vectors encoding the full-length human ErbB2 and ErbB3 sequences, pcDNAIII-ErbB2 and pcDNAIII-ErbB3, respectively, were provided by Dr. Kermit Carraway III. The expression vector pcDNAIII-PKA catalytic subunit was provided by Dr. J. Silvio Gutkind. Primary Cultures of Adult-derived Human Schwann Cells and Peripheral Nerve Fibroblasts—Human peripheral nerve tissue, consisting of nerve roots comprising the cauda equina, was provided by the Life Alliance Organ Recovery Agency at the University of Miami Miller School of Medicine. Nerve fragments were incubated 6–8 days at 37 °C in Dulbecco's Modified Eagle's Medium (DMEM, Invitrogen) containing 10% heat-inactivated fetal bovine serum (FBS, HyClone), and 2 μm forskolin and 10 nm neuregulin (D10/FN medium). For dissociation, connective tissue-free nerve fascicles were incubated in DMEM containing 10% FBS, 0.5 mg/ml collagenase type I (Worthington, Lakewood, NJ), and 2.5 mg/ml dispase II (Roche Applied Science) overnight at 37 °C. The resulting cell suspension was plated onto mouse laminin-coated tissue culture dishes in D10/FN medium and allowed to grow until confluency. SCs were resuspended and purified by immunopanning with monoclonal antibodies against primate NGFR p75 (hybridoma supernatant, ATCC). The purity of the cultures was 96–99% based on immunostaining with anti-S100 (Dako), a protein expressed specifically in SCs. Cells were used for experiments between 2 and 8 population doublings (1–3 passages). Fibroblasts from cauda equina were established by cultivating dissociated cell preparations on non-coated plastic dishes in DMEM containing 10% FBS without mitogens. The absence of neuregulin in the culture medium and an inappropriate substrate strongly retards the proliferation and survival of SCs, whereas the absence of forskolin allows fibroblasts to proliferate rapidly and override the growth of remaining SCs. Cultures obtained were >98% fibroblasts. [3H]Thymidine Incorporation Assays—Sub-confluent cultures of SCs growing on poly-l-Lysine (PLL)-laminin-coated 12-well plates (100,000 cells/well) or 24-well plates (50,000 cells/well) were deprived of mitogens for 2 days in DMEM-10% FBS and then for 1 day in HEPES-buffered DMEM containing 1% FBS. SCs return to quiescence following removal of the mitogenic stimulus for 3 days. The presence of a non-mitogenic concentration of FBS in the culture medium was essential to maintain cell attachment and prevent the loss of cells by apoptosis induced by serum removal. Cells were exposed to medium containing [3H]thymidine (0.25 μCi/ml) under the conditions described in the figure legends. As an ErbB/HER agonist, we used a neuregulin peptide consisting of the EGF homology domain of β1-heregulin (hereafter referred to as "neuregulin") that is sufficient to bind and activate ErbB2-ErbB3 heterodimers (23Barbacci E.G. Guarino B.C. Stroh J.G. Singleton D.H. Rosnack K.J. Moyer J.D. Andrews G.C. J. Biol. Chem. 1995; 270: 9585-9589Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 24Carraway K.L. 3rd, Soltoff S.P. Diamonti A.J. Cantley L.C. J. Biol. Chem. 1995; 270: 7111-7116Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar) and downstream signaling pathways in SCs (7Monje P.V. Bartlett Bunge M. Wood P.M. Glia. 2006; 53: 649-659Crossref PubMed Scopus (83) Google Scholar). Unless otherwise noted, a concentration of 10 nm neuregulin and 2 μm forskolin was used for all stimulation experiments. 48 h after stimulation, cells were washed three times with phosphate-buffered saline, lysed with 300 μl of Solvable™ and incorporated tritium in the cells was determined by liquid scintillation counting. Cultures were assayed in triplicate samples in each condition. Before lysis and tritium counting, cells were co-labeled with Cell Tracker Green (5-chloromethylfluorescein diacetate, Molecular Probes) and the nuclear dye Hoechst 33342 (Sigma) and monitored live by fluorescence microscopy. No indication of reduced metabolic activity or induced apoptotic nuclei was observed for any of the treatments. Cell Lines—HEK293T cells (ATCC) were routinely cultured in DMEM containing 10% FBS and penicillin-streptomycin-gentamycin (Invitrogen) on a PLL substrate. Transient Transfections—Cultures of HEK293T cells were allowed to grow to sub-confluency in 24-well PLL-coated dishes and then transfected overnight with the FuGENE 6 Reagent in DMEM-HEPES without FBS (0.25 μg of total plasmid DNA/well), according to the protocol suggested by the manufacturer (Roche Applied Science). The total amount of transfected DNA was made equivalent by adding the control plasmid, pcDNAIII-β-gal, an expression vector for the enzyme β-galactosidase. At 24 h post-transfection, medium was replaced and cells were subjected to stimulation followed by cell lysis and Western blotting analysis, as described below. Single cell suspensions (5 × 106 cells) of adult human SCs obtained from exponentially growing cultures were transfected using the Nucleofection method (4 μg of total plasmid DNA/transfection), according to the instructions suggested by the manufacturer (Oligodendrocyte Nucleofection Kit, program A-33, Amaxa Biosystems). After transfection, SCs were initially plated on PLL+laminin-coated six-well dishes in DMEM containing 10% FBS and then re-plated 4 h later to 24-well dishes (50,000 cells/well) for cell proliferation assays and Western blotting analysis. For experimentation, stimulation of transfected SCs was done 24-post transfection in HEPES-buffered DMEM containing 1% FBS. Transfection efficiency for SCs was 40–60% based on green fluorescent protein fluorescence from the expression vector pMAX-green fluorescent protein (Amaxa Biosystems), which was routinely used as a positive control to estimate transfection efficiency. Western Blots—Protein samples from total cell lysates were prepared by resuspending the cells in lysis buffer (50 mm Tris, 150 mm NaCl, 1% SDS, 0.5 mm dithiothreitol) containing protease inhibitors (1 mm phenylmethylsulfonyl fluoride, 20 μg/ml aprotinin, and 20 μg/ml leupeptin) and phosphatase inhibitors (phosphatase cocktails I and II, Sigma). Lysates were combined with SDS sample buffer (400 mm Tris/HCl, pH 6.8, 10% SDS, 50% glycerol, 500 mm dithiothreitol, 2 μg/ml bromphenol blue), followed by 10 min boiling. Protein aliquots were resolved in denaturing polyacrylamide gels (SDS-PAGE), and fractionated proteins were transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA). Membranes were blocked with 5% bovine serum albumin in Tris-Buffer saline (TBS) containing 0.05% Tween-20 (TBS-T) and incubated overnight with the appropriate dilution of each primary antibody (1:1000 unless otherwise noted). The membranes were washed three times with TBS-T prior to incubation with horse-radish peroxidase-conjugated secondary antibodies, 1:10,000 (Promega, Madison, WI). Immunoreactive protein bands were detected by enhanced chemiluminescence using ECL Advanced or ECL Plus, depending on signal intensity, according to the manufacturer's instructions (Amersham Biosciences). For densitometric analysis, films were scanned at high resolution using a Bio-Rad Fluor-S MultiImager, and the relative optic density of the protein bands was estimated using Quantity One software (Bio-Rad). Detection of PKA Activity—The kinase activity of PKA was assessed by immunodetecting the phosphorylation of cellular PKA substrates using an antibody that recognizes PKA-specific phospho-motifs ([RR]-X-[S*/T*]) in target proteins, either in fixed cells (immunostaining) or in cell lysates (Western blot). Curiously, phospho-PKA substrate antibodies detected only a limited set of substrates in cAMP-treated SCs, most likely due to the fact that only a proportion of the total cellular substrates phosphorylated by PKA, would be recognized by this specific antibody. Moreover, phosphorylated targets recognized by anti-PKA substrate antibodies were mostly cytoplasmic/membrane proteins based on immunostaining detection. For the immunostaining experiments, SCs were grown in 24-well plates coated sequentially with PLL and laminin and fixed with 4% paraformaldehyde (20 min, room temperature) followed by cold methanol (10 min, 4 °C). Cells were then washed three times with TBS and incubated with 5% normal goat serum in TBS (TBS-NGS) for 1 h at room temperature. Subsequently, the cells were incubated with anti-phospho-PKA substrate antibody (1:200 in TBS-NGS, overnight, 4 °C) and rinsed three times with TBS prior to incubation with anti-rabbit Alexa-594-conjugated secondary antibodies (1:200 in TBS-NGS, 1 h, room temperature). Cells were mounted with Vectashield containing 4′,6-diamidino-2-phenylindole (DAPI, Vector Laboratories, Burlingame, CA) and analyzed by conventional fluorescence microscopy. Digitalized images from immunofluorescence microscopy and from Western blot films were processed by using Adobe Photoshop version 7.0 and arranged for presentation using Adobe Illustrator CS3. In Vitro Kinase Assays—The phosphorylation of ErbB2 by PKA was assayed in vitro by following a standard PKA kinase assay, using a purified preparation of PKA catalytic subunit (Promega) and including purified GST-HER/ErbB2 679–1255 (human, recombinant, Calbiochem) as a substrate in the reaction mixture (PKA assay kit, Promega). The reactions were initiated by the addition of 1 μl of purified PKA and allowed to proceed for 30 min at room temperature before denaturing the proteins (10 min, boiling bath) and assaying the products by Western blot using anti-phospho PKA substrate and anti-phospho-ErbB2 Thr-686 antibodies. Reactions were carried out in the absence and presence of a specific PKA inhibitor peptide (PKI-[6–22]-NH2, Upstate Biotechnology, Lake Placid, NY), which highly discriminates between PKA and protein kinase C. ErbB/HER Sequence Analysis and Site-directed Mutagenesis— The available protein sequence of ErbB/HER2 from the different species analyzed was obtained from GenBank™ (NCBI), and the search for potential consensus PKA-specific motifs ([KR]-X-[T/S]) was done with the program PROSITE (free internet resource). Individual point mutation of Thr-686 on pcDNAIII-ErbB2 was generated by site-directed mutagenesis (BPS Bioscience, mutagenesis service). Mutagenesis primers were designed to introduce an ACG to GCG mutation (Thr to Ala) to generate an alanine replacement of Thr-686 in the full-length ErbB2 coding sequence. Rap1 Assays—To measure the nucleotide exchange activity of EPAC, we assayed the GTP-binding activity of its downstream effector Rap1, by using a standard method that involves the precipitation of GTP-bound Rap1 molecules by affinity binding to purified recombinant Ral-GDS-agarose beads (Rap1 activation assay, Roche Applied Science). SCs were allowed to grow up to 70–80% confluency in 10-cm plates coated with PLL-laminin (4 × 106 cells/dish), starved as described before and then stimulated with cAMP analogs for 30 min. The plates were washed with iced-cold TBS, and the cells were rapidly resuspended in lysis buffer. All subsequent steps were performed according to the manufacturer's recommendations. Purified samples containing GTP-bound Rap1 were denatured in SDS loading buffer, boiled, resolved by 15% SDS-PAGE, and analyzed by Western blotting using anti-total Rap1 antibodies (Santa Cruz Biotechnology, 1:1000). Samples from total cell lysates were analyzed in parallel as controls for total Rap1 expression. The Activation of PKA, but Not EPAC, Is Sufficient to Synergistically Enhance the Neuregulin-stimulated Proliferation of SCs—The mechanism underlying the action of cAMP in neuregulin-induced SC proliferation remains mostly undefined. As a first approach, we investigated the role of PKA or EPAC, by the use of pathway-selective cell-permeable cAMP analogs (25Rehmann H. "Experimental Procedures" Enzymol. 2005; 407: 159-173Google Scholar). We compared the effect of 6-Bnz-cAMP and 8-CPT-cAMP, two widely used PKA- and EPAC-selective cAMP analogs, respectively (26Enserink J.M. Christensen A.E. de Rooij J. van Triest M. Schwede F. Genieser H.G. Doskeland S.O. Blank J.L. Bos J.L. Nat. Cell Biol. 2002; 4: 901-906Crossref PubMed Scopus (617) Google Scholar), for their ability to regulate neuregulin/HER-dependent SC proliferation. By measuring the incorporation of [3H]thymidine in adult-derived human SCs, we observed that the selective activation of PKA by 6-Bnz-cAMP was sufficient to synergistically and dose dependently enhance neuregulin-stimulated S-phase entry, resembling the effects of forskolin, a direct activator of adenylyl cyclases (Fig. 1A) and non-selective analogs of cAMP, such as db-cAMP and 8-Br-cAMP (Fig. 1D). In contrast, when SCs were stimulated with neuregulin in the presence of 8-CPT-cAMP, neuregulin-stimulated S-phase entry was significantly reduced (Fig. 1B), without diminishing the metabolic activity or the survival of the cells (not shown). In addition, the activation of EPAC by 8-CPT-cAMP was sufficient to antagonize the synergistic action of 6-Bnz-cAMP and forskolin on neuregulin-induced S-phase entry (not shown). Controls for the relative potency and specificity of the cAMP analogs used in the experiments are shown in Fig. 1C. Similarly, the PKA-selective analogs 6-PHE-cAMP and 6-MB-cAMP synergistically enhanced, whereas the EPAC-selective analog PME-cAMP reduced, neuregulin-induced S-phase entry (Fig. 1D), supporting opposite roles of PKA and EPAC in mediating the effects of cAMP in SC proliferation. Consistent with these results and with the previously proposed role of PKA in cell cycle progression in SCs (27Kim H.A. DeClue J.E. Ratner N. J. Neurosci. Res. 1997; 49: 236-247Crossref PubMed Scopus (104) Google Scholar), pretreatment with H-89, a potent pharmacological inhibitor of PKA kinase activity (28Lochner A. Moolman J.A. Cardiovasc. Drug Rev. 2006; 24: 261-274Crossref PubMed Scopus (249) Google Scholar), dose dependently reduced the incorporation of [3H]thymidine in SCs stimulated with neuregulin in the presence of either forskolin or 6-Bnz-cAMP (Fig. 1E). Similarly, preincubation with either the inactive cAMP stereoisomer, Rp-cAMP, or with a cell-permeable active peptide from the heat-stable protein kinase inhibitor (myr-PKI), effectively antagonized the action of cAMP on SC proliferation (data not shown). To rule out a possible contribution of fibroblasts to the observed results, we determined the effect of cAMP on the proliferation of human fibroblasts from adult peripheral nerve. These fibroblasts were unresponsive to neuregulin (not shown), but they were responsive to fetal bovine serum (Fig. 1F) and to platelet-derived growth factor and fibroblast growth factor1 (not shown). As opposed to SCs, forskolin, but not 6-Bnz-cAMP, decreased the incorporation of [3H]thymidine induced by serum (Fig. 1F), platelet-derived growth factor and fibroblast growth factor1 (not shown), confirming the cell type specificity of cAMP action in SC cultures. Interestingly, stimulation with 8-CPT-cAMP was sufficient to dramatically reduce both serum- (Fig. 1F) and growth factor-induced (not shown) proliferation, further supporting a role of EPAC in mediating the anti-proliferative effects of cAMP in both SCs and fibroblasts. Overall, these results suggest that the regulation of S-phase progression by cAMP is not only cell type-specific but also highly dependent on the particular set of effectors activated by cAMP. Whereas EPAC activation converted cAMP from a proliferative to an anti-proliferative signal in SCs, the activation of PKA was by itself not only required but also sufficient to synergistically promote S-phase entry in the presence of neuregulin. cAMP Synergistically Enhances the Neuregulin-stimulated Tyrosine Phosphorylation of ErbB3 and the Activation of PI3K/Akt Signaling— cAMP signaling potentially converges at multiple levels in the signaling axes Ras-Raf-MEK-ERK (19Stork P.J. Schmitt J.M. Trends Cell Biol. 2002; 12: 258-266Abstract Full Text Full Text PDF PubMed Scopus (749) Google Scholar, 29Dumaz N. Marais R. FEBS J. 2005; 272: 3491-3504Crossref PubMed Scopus (256) Google Scholar) and PI3K-PDK-Akt (30Kim S. Jee K. Kim D. Koh H. Chung J. J. Biol. Chem. 2001; 276: 12864-12870Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 31Mei F.C. Qiao J. Tsygankova O.M. Meinkoth J.L. Quilliam L.A. Cheng X. J. Biol. 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