title: cAMP-GEF cytoprotection by Src tyrosine kinase activation of phosphoinositide-3-kinase p110 beta/alpha in rat hepatocytes.
abstract: Cyclic AMP protects against hepatocyte apoptosis by a protein kinase A-independent cAMP-GEF/phosphoinositide-3-kinase (PI3K)/Akt signaling pathway. However, the signaling pathway coupling cAMP-GEF with PI3K is unknown. The aim of this study was to investigate the role of Src tyrosine kinases (Src-TYK) and PI3K-p110 isoforms in this pathway. Studies were done in rat hepatocytes using the hydrophobic bile acid glycochenodeoxycholic acid (GCDC) to induce apoptosis. cAMP-binding guanine nucleotide exchange factors (cAMP-GEFs) were selectively activated by using 4-(4-chloro-phenylthio)-2'-O-methyladenosine-3'-5'-cyclic monophosphate (CPT-2-Me-cAMP), which sequentially phosphorylated Src-TYK (within 1 min) followed by Akt (within 5 min). The Src inhibitors PP2 and SU6656 inhibited basal and CPT-2-Me-cAMP-mediated Src and Akt phosphorylation. These inhibitors had no effect on CPT-2-Me-cAMP-mediated activation of Rap GTPases. CPT-2-Me-cAMP induced transient Src dependent autophosphorylation of the epidermal growth factor receptor (EGFR). Inhibition of the EGFR with AG 1478 partially inhibited the ability of CPT-2-Me to phosphorylate Akt. Whereas PP2 completely abolished the protective effect of CPT-2-Me-cAMP in GCDC induced apoptosis, AG 1478 partially inhibited the cytoprotective effect. CPT-2-Me-cAMP treatment resulted in Src-dependent activation of the p110 beta and alpha subunits of PI3K, but only the latter was sensitive to inhibition with AG 1478. In conclusion, activation of cAMP-GEFs results in phosphorylation of Src-TYK and Akt and activation of the p110 beta/alpha subunits of PI3K. Maximal cAMP-GEF-mediated Akt phosphorylation as well as protection from bile acid-induced apoptosis requires activation of Src-TYK and the EGFR. These studies support the existence of two pathways: cAMP-GEF/Rap/Src/PI3Kbeta/Akt and cAMP-GEF/Rap/Src/EGFR/PI3Kalpha/Akt, both of which are necessary for maximal cytoprotective effect of cAMP-GEFs in hepatocytes.
authors.affiliation: Department of Clinical Sciences, Tufts Cummings School of Veterinary Medicine, 200 Westboro Rd., North Grafton, MA 01536, USA.
authors.forename: Anna
authors.initials: A
authors.lastname: Gates
authors.forename: Simon
authors.initials: S
authors.lastname: Hohenester
authors.forename: M Sawkat
authors.initials: MS
authors.lastname: Anwer
authors.forename: Cynthia R L
authors.initials: CR
authors.lastname: Webster
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D058534:Class I Phosphatidylinositol 3-Kinases; D000242:Cyclic AMP / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000502:physiology; D005999:Glycochenodeoxycholic Acid / Q000494:pharmacology; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D022781:Hepatocytes / Q000187:drug effects* / Q000378:metabolism*; D007527:Isoenzymes; D008297:Male; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D015398:Signal Transduction; D019061:src-Family Kinases / Q000235:genetics / Q000378:metabolism*
chemical_list: D020662:Guanine Nucleotide Exchange Factors; D007527:Isoenzymes; D005999:Glycochenodeoxycholic Acid; D000242:Cyclic AMP; D019869:Phosphatidylinositol 3-Kinases; D058534:Class I Phosphatidylinositol 3-Kinases; C488371:p110delta protein, rat; D019061:src-Family Kinases