Charting calcium-regulated apoptosis pathways using chemical biology: role of calmodulin kinase II.
BACKGROUND
Intracellular free calcium ([Ca2+]i) is a key element in apoptotic signaling and a number of calcium-dependent apoptosis pathways have been described. We here used a chemical biology strategy to elucidate the relative importance of such different pathways.


RESULTS
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-kappaB 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.


CONCLUSIONS
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.
Olofsson
Maria Hägg
MH
Cancer Center Karolinska, Department of Oncology-Pathology, Karolinska Institute, S-171 76 Stockholm, Sweden.
Havelka
Aleksandra Mandic
AM
Cancer Center Karolinska, Department of Oncology-Pathology, Karolinska Institute, S-171 76 Stockholm, Sweden.
Brnjic
Slavica
S
Cancer Center Karolinska, Department of Oncology-Pathology, Karolinska Institute, S-171 76 Stockholm, Sweden.
Shoshan
Maria C
MC
Cancer Center Karolinska, Department of Oncology-Pathology, Karolinska Institute, S-171 76 Stockholm, Sweden.
Linder
Stig
S
Cancer Center Karolinska, Department of Oncology-Pathology, Karolinska Institute, S-171 76 Stockholm, Sweden.