Anti-apoptotic Signaling of the Insulin-like Growth Factor-I Receptor through Mitochondrial Translocation of c-Raf and Nedd4

Francesca Peruzzi,Marco Prisco,Andrea Morrione,Barbara Valentinis,Renato Baserga

doi:10.1074/jbc.m103188200

Francesca Peruzzi, Marco Prisco + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m103188200

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Abstract

The type 1 insulin-like growth factor receptor (IGF-IR) sends a strong anti-apoptotic signal by at least three different pathways. By using mutants of the IGF-IR, we showed that one of the pathways depends on residues of the IGF-IR (serines 1280--1283) that interact with 14.3.3 proteins. The result is the activation of Raf-1 and the mitochondrial translocation of both Raf-1 and Nedd4, a target of caspases. A mutant IGF-IR in which the serines at positions 1280--1283 have been mutated to alanine does not protect from apoptosis and fails to translocate Nedd4 or Raf-1 to the mitochondria. This failure is accompanied by a loss of cytochrome c from the mitochondria. The 14.3.3/Raf-1/Nedd4 pathway is operative in the presence or absence of the insulin receptor substrate-1.

Highlights

The mode of death is apoptosis, which has been repeatedly documented in previous papers from this and other laboratories [23, 25, 38, 39]. 32D IGF-IR cells survive in the absence of IL-3, if the medium is supplemented with IGF-I
Our results can be summarized as follows. 1) 32D cells expressing a wild type IGF-IR survive after IL-3 withdrawal, but they die if they express a receptor with a 4-serine mutations
2) There is a correlation between survival and mitochondrial translocation of both Raf-1 and Nedd4, a target of caspases [33]

Summary

EXPERIMENTAL PROCEDURES

Plasmids—The wild type IGF-IR and its mutants (4 serine and 6 serine mutants) are described by Romano et al [26] and by Peruzzi et al [10]. IGF-IR and IRS-1 Expression Levels—The levels of IGF-IR were monitored by Western blot, as described previously [10] using an antibody to the ␤-subunit of the IGF-IR (Santa Cruz Biotechnology). The antibody against IRS-1 was from Upstate Biotechnology, Inc. Detection of Mitochondrial Raf-1 and Nedd4 —Cells exponentially growing were washed five times with Hanks’ balanced salt solution and seeded (7 ϫ 106) in RPMI 1640 medium supplemented with 10% ⌬FBS only, supplemented with 10% ⌬FBS and 10% IL-3 (WEHI cell-conditioned medium), or with 10% ⌬FBS and 50 ng/ml IGF-I. After homogenization (25 strokes with a Dounce homogenizer B pestle), samples were centrifuged two times (at 2,500 ϫ g for 5 min) to remove the nuclei and centrifuged again at 13,000 ϫ g for 10 min to obtain the heavy membrane pellet This fraction was resuspended in buffer B and centrifuged again at 13,000 for 10 min. In Western blots, statistical significance between two measurements was determined by the twotailed Student’s t test analysis of variance

RESULTS

Percentage of differentiated cells

DISCUSSION