Outlines:

The cellular response to diverse external stimuli is controlled via a complex array of phosphorylation cascades. The ERK cascade is a prominent component of the MAP kinase family that, in particular, plays integral role in both growth factor and stress signalling. Recent studies identified members of the MAPK superfamily that include JNK/SAPK and p38 MAPK, which are activated by various environmental stresses such as UV irradiation or exposure to agents, in addition to classical MAPKs. Each MAPK group has a distinct substrate specificity and is regulated by a separate signal transduction pathway. Mammalian cells, therefore, contain multiple MAPK superfamily signal transduction pathways that mediate the effects of extracellular stimuli on a wide array of biological processes. The activities of these various MAPKs can be measured directly in acrylamide gel containing various substrates.
Cells (1 x 106) treated/untreated with various drugs were lysed in lysis buffer and the protein concentration was determined. To assay total cell lysate, equal amounts of protein (50-100 ug) were electrophoresed in 10 % SDS-PAGE containing either MBP (0.5 mg/ml), GST-c-Jun (1-79; 0.1 mg/ml), or GST-ATF2 (0.1 mg/ml) as a substrate. After electrophoresis, SDS was removed from the gel, protein was renatured, and a kinase assay was carried out by incubating the gel in buffer containing [gamma-32P]ATP. Gels were washed and dried, and the incorporated radio activity was analyzed by autoradiography.

Cylolrienin A activates a kinase with an apparent molecular mass of 36 kDa with kinetics different from JNK/SAPK and p38 MAPK in HL-60 cells. (A) in-gel kinase activity of cells treated with cytotrienin A. Cells were incubated with cytotrienin A at the concentrations indicated. Cell lysates were eleclrophoresed in 10% SDS-PAGE, and kinase activity was determined by an in-gel kinase assay using MBP as a substrate. (B) kinetics of the activation of p.36 MBP kinase. JNK/SAPK. and p.38 MAPK during apoptosis induced by cytotrienin A. Cells were incubated with 100 ng/ml cytotrienin A for the times indicated, and the activation of the JNK/SAPK and p38 MAPK was analyzed by Western blotting using antibodies that specifically recognize the activated JNK/SAPK and p38 MAPK kinases. The p36 MBP kinase activity was measured as described in A. (C) effect of a specific inhibitor of p38 MAPK, SB203580, on p36 MBP kinase activation. Cells were treated with 100 ng/ml cytotrienin A in the presence of SB203580. After incubating for 2 h, p38 MAPK activation and p36 MBP kinase activity were measured as described in B. (Ref:1)

References:

1. H. Kakeya, R. Onose, H. Osada.
   Caspase-mediated activation of a 36-kDa myelin basic protein kinase during anticancer drug-induced apoptosis.
   Cancer Res., 58, 4888- 4894 (1998).