Cell-type-specific activation of p38 protein kinase cascades by the novel tumor promoter palytoxin

Palytoxin is a potent non-12-O-tetradecanoylphorbol-13-acetate (TPA)- type skin tumor promoter. We used COS7 and HeLa cells to investigate the protein kinase cascades by which palytoxin activates the mitogen-activated protein kinase (MAPK) p38. Three p38 kinases have been identified: stress- activated protein kinase/extracellular signal-regulated kinase kinase-1 (SEK1), MAPK kinase 3 (MKK3), and MKK6. SEK1 phosphorylates and activates both p38 and c-Jun NH₂-terminal kinase (JNK), whereas MKK3 and MKK6 selectively phosphorylate and activate p38. Although transiently overexpressed SEK1 activates p38 in cells, the importance of endogenous SEK1 for the activation of p38 by specific types of stimuli is unclear because some agents, such as sorbitol, can activate p38 in cells derived from SEK1 knockout mice. Because we previously showed that palytoxin activates JNK through an SEK1-dependent pathway, we investigated whether SEK1 also mediates the activation of p38 by palytoxin. The results presented here demonstrate that endogenous SEK1 does play an important role in the activation of p38 by palytoxin in specific cell types. In COS7 cells, palytoxin stimulated the phosphorylation of SEK1 and MKK6, and expression of dominant negative mutants of either SEK1 or MKK6 inhibited palytoxin-stimulated p38 activation. In HeLa cells, palytoxin stimulated the phosphorylation of MKK3 in addition to SEK1 and MKK6. In contrast to COS7 cells, in HeLa cells expression of a dominant negative mutant of SEK1 did not inhibit palytoxin-stimulated activation of p38, although expression of dominant negative mutants of either MKK3 or MKK6 did inhibit palytoxin-stimulated p38 activation in this cell type. These studies indicate that the importance of SEK1 in the activation of p38 by palytoxin depends on the ability of palytoxin to activate MKK3 and MKK6.