Cytochrome c and IP 3 R: A Deadly Handshake

Abstract

The mitochondria and endoplasmic reticulum (ER) are both involved in the apoptosis pathway, and release of calcium from the ER contributes to this process of cell death (see Mattson and Chan). ER calcium release is also involved in many nonpathological signaling processes, and Boehning et al. show that the interaction of cytochrome c (cyt c) released from mitochondria with the inositol trisphosphate receptors (IP 3 Rs) of the ER may be how the ER calcium release can reach pathologic levels. An interaction between cyt c and the IP 3 R was identified in a yeast two-hybrid screen, and these two proteins coimmunoprecipitated from cells treated with the broad-spectrum kinase inhibitor STS, which induces apoptosis. Furthermore, cell fractionation studies of cells treated with STS showed that cyt c accumulated in the ER fraction and was depleted from the mitochondrial fraction. Movement of cyt c to the ER fraction was not inhibited by exposure of the cells to caspase inhibitors, which suggests that translocation of cyt c occurred before caspase activation during apoptosis. Fluorescence resonance energy transfer assays indicated that cyt c and IP 3 Rs come into close association after induction of apoptosis with STS. IP 3 Rs are sensitive to calcium such that at concentrations in the nanomolar range (resting levels in a cell), calcium stimulates IP 3 R calcium release. In contrast, at higher micromolar concentrations of calcium, IP 3 R-induced calcium release is inhibited by calcium. Microsomal preparations exposed to cyt c and micromolar concentrations of calcium did not exhibit calcium-induced inhibition of IP 3 R calcium release. In vivo calcium imaging suggested that a spike in calcium precedes cyt c release from the mitochondria and then calcium levels remain elevated. Expression of the cyt c-binding domain of the IP 3 R acted as a dominant negative, preventing calcium oscillations and cyt c release in response to STS. Thus, release of small amounts of cyt c leads to an interaction with the IP 3 R on the ER, preventing calcium inhibition of ER calcium release. The resulting global increase in calcium triggers massive release of cyt c, which then acts in the positive feedback loop to maintain ER calcium release through the IP 3 Rs. M. P. Mattson, S. L. Chan, Calcium orchestrates apoptosis. Nat. Cell Biol. 5 , 1041-1043 (2003). [Online Journal] D. Boehning, L. Patterson, L. Sadaghat, N. O. Blebova, T. Kurosaki, S. H. Snyder, Cytochrome c binds to inositol (1,4,5) trisphosphate receptors, amplifying calcium-dependent apoptosis. Nat. Cell Biol. 5 , 1051-1061 (2003). [Online Journal]