Cytotoxic T cells (CTLs) kill target cells by releasing lytic agents via regulated exocytosis. Three signals are known to be required for exocytosis: an increase in intracellular Ca2+, activation of protein kinase C (PKC) and activation of extracellular signal regulated signal kinase (ERK). ERK activation required for exocytosis depends on activity of PKC. The simplest possibility is that the sole effect of PKC required for exocytosis is ERK activation. Testing this requires dissociating ERK and PKC activation. We did this using TCR-independent stimulation of TALL-104 human leukemic CTLs. When cells are stimulated with thapsigargin and PMA, agents that increase intracellular Ca2+ and activate PKC, respectively, PKC-dependent ERK activation is required for lytic granule exocytosis. Expressing a constitutively active mutant MAP kinase kinase activates ERK independent of PKC. However, activating ERK without PKC does not support lyric granule exocytosis, indicating that there are multiple effects of PKC required for granule exocytosis. (C) 2008 Elsevier Inc. All rights reserved.