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Protection from rapamycin-induced apoptosis by insulin-like growth factor-I is partially dependent on protein kinase

Kuntebommanahalli N Thimmaiah1, John B Easton, Peter J Houghton

  • 1Department of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.

Cancer Research
|February 25, 2010
PubMed

Insights

Insulin-like growth factor-I (IGF-I) prevents rapamycin-induced apoptosis in sarcoma cells by phosphorylating Bad via phosphoinositide 3-kinase (PI3K) and protein kinase C (PKC) pathways, independent of Ras-ERK and Akt signaling.

Area of Science:

  • Cellular Biology
  • Molecular Oncology
  • Signal Transduction

Background:

  • Rapamycin is a drug used in cancer therapy that induces apoptosis (programmed cell death) in sarcoma cells.
  • Insulin-like growth factor-I (IGF-I) is known to promote cell survival and can counteract apoptosis induced by various stimuli.
  • The precise molecular mechanisms by which IGF-I inhibits rapamycin-induced apoptosis in sarcoma cells remain incompletely understood.

Purpose of the Study:

  • To elucidate the signaling pathway utilized by IGF-I to inhibit rapamycin-induced apoptosis in sarcoma cells.
  • To identify the key proteins and phosphorylation sites involved in this protective signaling cascade.
  • To determine the role of Bad protein and its phosphorylation in mediating IGF-I's anti-apoptotic effect.

Main Methods:

  • Utilized gene knockdown techniques with small interfering RNAs (siRNAs) to silence specific genes (Bad, Bid, Akt1, Akt2, PKC isoforms).
  • Employed in vitro kinase assays and immunoprecipitation to assess protein-protein interactions and phosphorylation events.
  • Generated and tested cells overexpressing wild-type Bad or mutated Bad proteins (single, double, or triple phosphorylation site mutants) to confirm functional roles.

Main Results:

  • IGF-I inhibits rapamycin-induced apoptosis through a pathway independent of Ras-ERK and Akt signaling.
  • IGF-I-mediated Bad phosphorylation at Ser112, Ser136, and Ser155 requires phosphoinositide 3-kinase (PI3K) and specific protein kinase C (PKC) isoforms (mu, epsilon, theta).
  • Phosphorylated Bad is sequestered in the cytosol by 14-3-3gamma, preventing its mitochondrial localization and subsequent pro-apoptotic signaling. PI3K activation is necessary for PKC activation, which directly phosphorylates Bad.

Conclusions:

  • IGF-I protects sarcoma cells from rapamycin-induced apoptosis by activating a PI3K- and PKC-dependent pathway that phosphorylates the pro-apoptotic protein Bad.
  • Multiple phosphorylation sites on Bad are crucial for IGF-I's protective effect, leading to its cytosolic sequestration.
  • This study identifies a novel signaling axis involving IGF-I, PI3K, PKCs, and Bad phosphorylation as a key mechanism for promoting sarcoma cell survival against rapamycin treatment.

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