AbstractWe recently demonstrated the activation of phosphatidylinositol 3‐kinase (PI3‐K/Akt) survival pathway in Jurkat T leukemia cells known for their sensitivity to the tumor necrosis factor (TNF)‐related apoptosis‐inducing ligand (TRAIL)/Apo2L cytotoxic action. The present investigation was done to elucidate the role of cAMP‐response element‐binding (CREB) protein in this system. Jurkat T cells were treated with 100–1,000 ng/ml TRAIL for time intervals up to 24 h in the presence or absence of selective pharmacologic inhibitors of PI3‐K/Akt (LY294002) or p38 MAPK (SB253580) pathways. Upon TRAIL treatment, a dose‐dependent increase in the percentage of apoptotic cells as well as in caspase‐3 activity was observed. A further enhancement of apoptotic cell death was obtained with the use of CREB1 siRNA technology, as demonstrated by flow cytometry. Western blot analysis showed a high constitutive level of CREB phosphorylation at Ser133 in Jurkat T cells under normal serum culture conditions. Under low serum culture conditions, an early (within 1 h) and transient increase in CREB phosphorylation was detected in response to both TRAIL doses and reduced upon pre‐treatment with LY294002 or SB253580, demonstrating the PI3‐K/Akt‐ and p38 MAPK‐dependency of this effect. The parallel analysis in immune fluorescence demonstrated the nuclear translocation of the phosphorylated form upon treatment with 100 ng/ml TRAIL, whereas the immune labeling was mainly detectable in the cytoplasm compartment upon the higher more cytotoxic dose. These results let us hypothesize that CREB activation can be an important player in the complex cross‐talk among pro‐ and anti‐apoptotic pathways in this peculiar cell model. J. Cell. Physiol. 214:192–200, 2008. © 2007 Wiley‐Liss, Inc.