Akt kinase reducing endoplasmic reticulum Ca2+ release protects cells from Ca2+-dependent apoptotic stimuli

Abstract

The proto-oncogene Akt is a potent inhibitor of apoptosis, and it is activated in many human cancers. A number of recent studies have highlighted the importance of the inositol 1,4,5-trisphosphate (IP3) receptor(IP3R) in mediating calcium (Ca2+) transfer from the endoplasmic reticulum (ER) to the mitochondriain several models of apoptosis. Akt is a serine-threonine kinase and recent data indicate the IP3R as a target of its phosphorylation activity. Here we show that HeLa cells, overexpressing the constitutively active myristoylated/palmitylatedAKT1 (m/p-AKT1), were found to have a reduced Ca2+ release from ER after stimulation with agonist coupled to the generation of IP3. In turn, this affected cytosolic and mitochondria Ca2+ response after Ca2+release from the ER induced either by agonist stimulation or by apoptotic stimuli releasing Ca2+ from intracellular stores. Most importantly, this alteration of ER Ca2+ content and release, reduces significantly cellular sensitivity to Ca2+ mediated proapoptotic stimulation. These results reveal a primary role of Akt in shaping intracellular Ca2+ homeostasis, that may underlie its protective role against some proapoptotic stimuli.

Fig. 1. Modifications of ER Ca²⁺ kinetic releases by overexpression of Akt in HeLa cells

(A) Effects of m/p-AKT1 on the ER Ca²⁺ concentration ([Ca²⁺]_er) in control and in m/pAKT1-overexpressing HeLa cells. (B) Reduced ER Ca²⁺ release in m/pAKT1-overexpressing cells when stimulated with 100µM histamine. To induce Ca²⁺ release from ER, the cells were challenged with an agonist that, trough interaction with G protein coupled receptors, evokes a rapid discharge from inositol 1,4,5 phosphate receptors (IP3Rs). The bars in the graph show the extent of the reduction in the mean rate of Ca²⁺ released induced by overexpressing m/p-AKT1 after cell stimulation, both for the first 50 seconds (upper graph) and for the maximum rate (lower graph). (C) HeLa cells were transfected with m/p-AKT1 or mock transfected in control cells. Western blot analysis shows that overexpression of constitutively active AKT does not affect the expression of different types of IP3 receptors.

Fig. 2. Alteration of cytosolic and mitochondrial Ca²⁺ homeostasis in Akt-overexpressing cells

(A) Cytosolic Ca²⁺ homeostasis in control and m/pAKT1-overexpressing HeLa cells. Where indicated, the cells were stimulated with 100µM histamine. (B) Mitochondrial Ca²⁺ homeostasis in control and m/pAKT1-overexpressing HeLa cells. Where indicated, the cells were stimulated with 100µM histamine.

Fig. 3. Effect of Akt on cell death induced by Ca²⁺-dependent apoptotic stimuli

(A) HeLa cells were co-transfected with mtGFP and m/p-AKT1 or mtGFP alone in control cells. At 36 h post-transfection, cells were treated with arachidonic acid (80 µM) and H₂O₂ (1 mM) for 4 hours. The data show the change of percentage of GFP fluorescent cells among the whole cell population (determined by phase contrast microscopy), averaging values obtained by analyzing more than 50 fields. (B) HeLa cells were transfected and treated at the same manner of Fig. A, and then aliquots of cells were centrifuged and lysates were assayed for caspase-3 activity as described in “Experimental Procedures”. The upper panel shows representative traces of a singular experiment. The data are the mean of different angular coefficients ± S.E. of five independent experiments; the bars in the graph (lower panel) show the change of percentage of caspase-3 activation compared to untreated cells.

Fig. 4. Akt reduces [Ca²⁺]_c elevation generated by H₂O₂ and arachidonic acid

Effects of m/p-AKT1 on [Ca²⁺]_c increase induced by H₂O₂ (A) and arachidonic acid (B). HeLa cells were loaded with the Ca²⁺ indicator Fura-2/AM and [Ca²⁺]_c changes were measured as details in Materials and Methods. The coverslips with the cells were maintained in 1mM Ca²⁺/KRB and, where indicated, the cells were challenged with 1 mM H₂O₂ or 80 µM arachidonic acid. The traces show the calibrated [Ca²⁺]_c values. Experiments were repeated at least 5 times.