CASPASE-MEDIATED ACTIVATION OF A 36-KDA MYELIN BASIC-PROTEIN KINASE DURING ANTICANCER DRUG-INDUCED APOPTOSIS

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 pr ocess, we performed an in-gel kinase assay using myelin basic protein (MBP) as a substrate and found the activation of kinase with an appare nt molecular mass of 36 kDa (p36 MBP kinase). The dose of cytotrienin A required to activate p36 MBP kinase was consistent,vith that require d to induce apoptotic DNA fragmentation in HL-60 cells. This p36 MBP k inase 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 w as immunologically different from MAPK superfamily molecules such as E RK1, JNK isoforms, and p38 MAPK. In addition, the p36 MBP kinase activ ation and apoptotic DNA fragmentation were inhibited by antioxidants s uch as N-acetylcysteine and reduced-form glutathione. The p36 MBP kina se activation was also observed during hydrogen peroxide (H2O2) and ok adaic acid-induced apoptosis. Although a specific inhibitor of caspase -3-like proteases (Ac-DEVD-CHO) or a specific inhibitor of caspase-1-l ike proteases (Ac-YVAD-CHO) did not block the cytotrienin A-, H2O2-, o r okadaic acid-induced apoptosis, a broad specificity inhibitor of cas pases (Z-Asp-CH2-DCB) strongly inhibited the apoptosis of HL-60 cells. Surprisingly, Z-Asp-CH2-DCB inhibited the activation of p36 MBP kinas e 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 oxyg en species could be included in the apoptotic events. Moreover, accord ing to the Western blotting using the antibodies against MST1/Krs2 or MST2/Krs1, it is suggested that the p36 MBP kinase is an active proteo lytic 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 p athways leading to apoptosis, and controlling this p36 MBP kinase path way might he a novel strategy for cancer chemotherapy.