A novel anticancer
drug,
cytotrienin A, isolated from Streptomyces sp., induces apoptosis (or programmed cell death) in human promyelocytic
leukemia HL-60 cells within 4 h. To elucidate the mechanism of this process, we performed an in-gel
kinase assay using
myelin basic protein (MBP) as a substrate and found the activation of
kinase with an apparent molecular mass of 36 kDa (p36 MBP
kinase). The dose of
cytotrienin A required to activate p36 MBP
kinase was consistent with that required to induce apoptotic DNA fragmentation in HL-60 cells. This p36 MBP
kinase was activated with kinetics distinct from the activation of JNK (
c-Jun N-terminal kinase)/stress-activated
protein kinase and
p38 MAPK (
mitogen-activated protein kinase). Importantly, the p36 MBP
kinase was immunologically different from MAPK superfamily molecules such as ERK1, JNK
isoforms, and
p38 MAPK. In addition, the p36 MBP
kinase activation and apoptotic DNA fragmentation were inhibited by
antioxidants such as
N-acetylcysteine and reduced-form
glutathione. The p36 MBP
kinase activation was also observed during
hydrogen peroxide (H2O2) and
okadaic acid-induced apoptosis. Although a specific inhibitor of caspase-3-like
proteases (
Ac-DEVD-CHO) or a specific inhibitor of caspase-1-like
proteases (
Ac-YVAD-CHO) did not block the
cytotrienin A-, H2O2-, or
okadaic acid-induced apoptosis, a broad specificity inhibitor of
caspases (Z-Asp-CH2-DCB) strongly inhibited the apoptosis of HL-60 cells. Surprisingly,
Z-Asp-CH2-DCB inhibited the activation of p36 MBP
kinase induced by
cytotrienin A or H2O2, but did not inhibit the activation of JNK/stress-activated
protein kinase and
p38 MAPK. Taken together, these results indicate that p36 MBP
kinase activation is downstream of the activation of Z-Asp-CH2-DCB-sensitive
caspases, and
reactive oxygen species could be included in the apoptotic events. Moreover, according to the Western blotting using the
antibodies against MST1/Krs2 or MST2/Krs1, it is suggested that the p36 MBP
kinase is an active proteolytic product of MST1/Krs2 and MST2/Krs1, which are originally cloned by virtue of its homology to the budding yeast Ste20
kinase. Thus, the p36 MBP
kinase might be a common component of the diverse signaling pathways leading to apoptosis, and controlling this p36 MBP
kinase pathway might be a novel strategy for
cancer chemotherapy.