Charting calcium-regulated apoptosis pathways using chemical biology: role of calmodulin kinase II

A set of 40 agents ("bioprobes") that induce apoptosis was first identified by screening of a chemical library. Using p53, AP-1, NFAT and NF-κB reporter cell lines, these bioprobes were verified to induce different patterns of signaling. Experiments using the calcium chelator BAPTA-AM showed that Ca2+ was involved in induction of apoptosis by the majority of the bioprobes and that Ca2+ was in general required several hours into the apoptosis process. Further studies showed that the calmodulin pathway was an important mediator of the apoptotic response. Inhibition of calmodulin kinase II (CaMKII) resulted in more effective inhibition of apoptosis compared to inhibition of calpain, calcineurin/PP2B or DAP kinase. We used one of the bioprobes, the plant alkaloid helenalin, to study the role of CaMKII in apoptosis. Helenalin induced CaMKII, ASK1 and Jun-N-terminal kinase (JNK) activity, and inhibition of these kinases inhibited apoptosis.Our study shows that calcium signaling is generally not an early event during the apoptosis process and suggests that a CaMKII/ASK1 signaling mechanism is important for sustained JNK activation and apoptosis by some types of stimuli.Calcium (Ca2+) is a universal signaling molecule regulating many aspects of cellular function and is one of the key elements of apoptotic signaling pathways [1]. Some agents, including glucocorticoids, the endoplasmic reticulum (ER) Ca2+-ATPase inhibitor thapsigargin and various cancer therapeutic drugs, mobilize Ca2+ stores and trigger apoptosis by early transient elevation of intracellular free calcium ([Ca2+]i). However, elevation of [Ca2+]i has been described also at later stages of the apoptotic process [1]. A number of calcium-mediated apoptosis signaling mechanisms have been described. Mitochondria are located in microdomains close to Ca2+ channels of the ER where cytosolic Ca2+ concentrations may become high on channel opening [2]. Mitochondrial Ca2+ uptake may result in mitochondrial permeability tr