Mitogen activation of human peripheral T lymphocytes induces the formation of new cyclic AMP response element-binding protein nuclear complexes

A large body of evidence indicates that experimental agents which raise cellular cAMP levels inhibit T cell growth and division. By contrast, many studies have reported that mitogen activation of T cells increases cAMP levels, implying a positive physiological role for cAMP in the activation process. In the present study we demonstrate that mitogen activation of human peripheral T lymphocytes induces nuclear factors that form complexes with cyclic AMP response element-binding protein (CREB). Four complexes are identified by the electrophoretic mobility shift assay, two of which are induced by mitogen activation. All four complexes contain CREB and are bound to the cAMP response element (CRE) core sequence (TGACGTCA), as indicated by antibody and oligonucleotide competition experiments. Binding of the four complexes to CRE is prevented by dephosphorylation of nuclear extracts and is restored by rephosphorylation with cAMP-dependent protein kinase or endogenous kinases. Similar complexes are detected in nuclear extracts of Jurkat cells. Mitogen induction of the electrophoretic mobility shift assay complexes is not accounted for by protein phosphorylation or by induction of CREB. Rather, the data indicate that mitogen increases the levels of a nuclear factor(s) that dimerizes with CREB. Induction of new CREB complexes implies a physiological role for cAMP in mitogen activation of T lymphocytes.     

The mouse elongation factor-2 gene: isolation and characterization of the promoter

Elongation factor 2 (EF-2) is a protein involved in peptide chain elongation in eukaryotes. We isolated the mouse EF-2 gene and characterized its promoter. We showed that the majority of enhancer elements were located within 500 bp of the flanking sequence and identified a factor binding site sequence (CGTCACGTGACGC) located between nucleotides -58 and -47 containing two CGTCA motifs separated by two nucleotides. The motif represents a half-site for binding of the cAMP response element (CRE) binding protein (CREB). Mutation analysis indicated that the presence of one CGTCA site alone conferred cAMP inducibility, but the presence of one or two CGTCA sites and spacing nucleotides elicited cAMP-independent, constitutive expression. UV cross-linking and DNA affinity chromatography revealed that three 40-, 43-, and 65-kD proteins bound to the CRE-like element. Of these, the 65-kD protein was unique to the CRE-like element. The 40-kD protein was ATF1 and the 43-kD protein with the molecular size of CREB was not CREB, on the basis of reactivity to their respective antibodies. Because ATF1 responds poorly to cAMP induction, it is likely the contributor to the constitutive expression rather than inductive expression of the CRE-like element, and, thus, the EF-2 gene.