In this study we report the activation of c-Jun Nterminal kinase (JNK) in human K562 erythroleukemia cells undergoing hemin-mediated erythroid differentiation, which occurs concomitantly with activation of heat shock factor 2 (HSF2) and leads to a simultaneous in vivo phosphorylation of c-Jun. The activation of JNK occurs through activation of mitogen-activated protein kinase kinase (MKK) 4 and not by activation of MKK7 or inhibition of JNK-directed phosphatases. We have previously shown that overexpression of the HSF2-L L isoform inhibits the activation of HSF2 upon hemin-induced erythroid differentiation. Here we demonstrate that HSF2-L L overexpression blocks the hemin-induced activation of the MKK4-JNK pathway, suggesting an erythroid lineage-specific JNK activation likely to be regulated by HSF2. ß