Co-stimulation of human peripheral blood mononuclear cells with IL-2 and anti-CD3 monoclonal antibodies induces phosphorylation of CREB.
Phosphorylation of the cAMP-response element binding protein CREB within 1 h of CD2 but not CD3 cross-linking of human PBMC was recently demonstrated. The absence of P-CREB following CD3 cross-linking was unexpected, as other laboratories reported increased phosphorylation of CREB following CD3 cross-linking of the Jurkat lymphocyte cell line. Due to Jurkat T-cells being IL-2-independent, it was postulated that IL-2 might provide a necessary co-stimulus for phosphorylation of CREB in primary lymphocytes. Therefore, P-CREB was evaluated following co-stimulation of human PBMC through the IL-2 and CD2 or CD3 receptors. IL-2 did not further augment phosphorylation of CREB following CD2 cross-linking. However, while neither IL-2 nor CD3 cross-linking alone induced P-CREB, a 4.5-fold increase in phosphorylation of CREB within 1 h of IL-2/CD3 co-stimulation was observed. Phosphorylation was not associated with the induction of cAMP, and inhibition of PKA signaling had no effect on P-CREB. Consistent with signal transduction through p56lck or p59fyn, inhibition of PTK signaling reduced phosphorylation 50%. Interestingly, inhibiting PKC signaling with calphostin C further increased P-CREB levels 3-fold over that observed in IL-2/CD3 co-stimulated cells not pretreated with a PKC inhibitor. In contrast to previous studies performed in the absence of exogenous IL-2, no increase in binding of CREB to a 32P-labeled oligonucleotide probe was observed by electrophoretic mobility shift assay. These data suggest that the IL-2 and CD3 signaling pathways provide a necessary and co-operative stimulus promoting phosphorylation of CREB following receptor cross-linking.
Guyot
D J
DJ
Center for Retrovirus Research and Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus 43210-1093, USA.
Newbound
G C
GC
Lairmore
M D
MD
D000911:Antibodies, Monoclonal / Q000494:pharmacology; D018801:CD2 Antigens / Q000276:immunology; D017252:CD3 Complex / Q000276:immunology*; D000242:Cyclic AMP / Q000494:pharmacology; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D004247:DNA / Q000235:genetics; D006801:Humans; D007376:Interleukin-2 / Q000494:pharmacology*; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000187:drug effects*; D008213:Lymphocyte Activation / Q000187:drug effects; D015345:Oligonucleotide Probes / Q000187:drug effects; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000494:pharmacology; D017260:Receptor-CD3 Complex, Antigen, T-Cell / Q000502:physiology; D015375:Receptors, Interleukin-2 / Q000502:physiology; D012694:Serine / Q000378:metabolism; D015398:Signal Transduction; D013936:Thymidine / Q000378:metabolism
D000911:Antibodies, Monoclonal; D018801:CD2 Antigens; D017252:CD3 Complex; D017362:Cyclic AMP Response Element-Binding Protein; D007376:Interleukin-2; D015345:Oligonucleotide Probes; D047428:Protein Kinase Inhibitors; D017260:Receptor-CD3 Complex, Antigen, T-Cell; D015375:Receptors, Interleukin-2; D012694:Serine; D004247:DNA; D000242:Cyclic AMP; D011494:Protein Kinases; D013936:Thymidine