title: Protection from rapamycin-induced apoptosis by insulin-like growth factor-I is partially dependent on protein kinase C signaling.
abstract: Rapamycin-induced apoptosis in sarcoma cells is inhibited by insulin-like growth factor-I (IGF-I) through a signaling pathway independent of Ras-extracellular signal-regulated kinase 1/2 and Akt. IGF-I induces Bad phosphorylation (Ser112, Ser136, and Ser155) in a pathway involving phosphoinositide 3' kinase (PI3K) and protein kinase C (PKC; mu, epsilon, or theta) resulting in sequestering Bad from mitochondria and subsequently interacting with 14-3-3gamma in the cytosol. Gene knockdown of Bad, Bid, Akt1, Akt2, PKC-mu, PKC-epsilon, or PKC-theta was achieved by transient transfection using small interfering RNAs. Results indicate that IGF-I signaling to Bad requires activation of PI3K and PKC (mu, theta, epsilon) but not mTOR, Ras-extracellular signal-regulated kinase 1/2, protein kinase A, or p90(RSK). Wortmannin blocked the phosphorylation of PKC-mu (Ser744/Ser748), suggesting that PI3K is required for the activation of PKCs. PKCs phosphorylate Bad under in vitro conditions, and the association of phosphorylated Bad with PKC-mu or PKC-epsilon, as shown by immunoprecipitation, indicated direct involvement of PKCs in Bad phosphorylation. To confirm these results, cells overexpressing pEGFP-N1, wt-Bad, or Bad with a single site mutated (Ser112Ala; Ser136Ala; Ser155Ala), two sites mutated (Ser(112/136)Ala; Ser(112/155)Ala; Ser(136/155)Ala), or the triple mutant were tested. IGF-I protected completely against rapamycin-induced apoptosis in cells overexpressing wt-Bad and mutants having either one or two sites of phosphorylation mutated. Knockdown of Bid using small interfering RNA showed that Bid is not required for rapamycin-induced cell death. Collectively, these data suggest that IGF-I-induced phosphorylation of Bad at multiple sites via a pathway involving PI3K and PKCs is important for protecting sarcoma cells from rapamycin-induced apoptosis.
authors.affiliation: Department of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.
authors.forename: Kuntebommanahalli N
authors.initials: KN
authors.lastname: Thimmaiah
authors.forename: John B
authors.initials: JB
authors.lastname: Easton
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Houghton
mesh_terms: D048948:14-3-3 Proteins / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D045744:Cell Line, Tumor; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000494:pharmacology; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology*; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis; D012208:Rhabdomyosarcoma / Q000201:enzymology / Q000473:pathology; D020123:Sirolimus / Q000494:pharmacology*; D058570:TOR Serine-Threonine Kinases; D051025:bcl-Associated Death Protein / Q000378:metabolism
chemical_list: D048948:14-3-3 Proteins; D017868:Cyclic AMP-Dependent Protein Kinases; D004815:Epidermal Growth Factor; D007328:Insulin; D007334:Insulin-Like Growth Factor I; D047908:Intracellular Signaling Peptides and Proteins; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D019869:Phosphatidylinositol 3-Kinases; D010982:Platelet-Derived Growth Factor; D011493:Protein Kinase C; D017346:Protein Serine-Threonine Kinases; D019253:Proto-Oncogene Proteins c-bcl-2; D020123:Sirolimus; D058570:TOR Serine-Threonine Kinases; D051025:bcl-Associated Death Protein; C494819:BAD protein, human; C546842:MTOR protein, human