Temporal and Subunit-specific Modulations of the Rel/NF-κB Transcription Factors Through CD28 Costimulation

Abstract

Stimulation of highly purified primary T lymphocytes through CD2 and CD28 adhesion molecules induces a long-term proliferation, dependent on persistent autocrine secretion of interleukin 2 (IL-2), high and prolonged expression of inducible CD25/IL-2 receptor ␣ chain (IL-2R␣), and secretion of growth factors such as the granulocyte-macrophage colony-stimulating factor (GM-CSF). CD28 costimulation appears to activate cytokine gene expression through conserved B-related CD28 response (CD28RE) or cytokine 1 (CK-1) elements in addition to canonical NF-Bbinding sites. In this report, we assess: 1) the evolution of the expression, over an 8-day time period, of the Rel/NF-B family of proteins in costimulated versus TcR/CD3-stimulated primary T cells; 2) the impact of changes on the in vitro occupancy of GM-CSF B and CK-1, as well as IL-2R␣ B sites; and 3) the differential regulation of newly synthesized p65 and c-Rel by IB proteins. We show that CD2؉CD28 stimulation specifically induces, at maximal T cell proliferation phase, sustained nuclear overexpression of NFKB2 p52 and c-Rel subunits which might rely on long-lasting processing of p100 precursor for p52 and increased neosynthesis of c-Rel. This up-regulation correlates with sustained occupancy of GM-CSF B and CK-1 elements by both proteins. Conversely, these subunits do not appear to bind to the IL-2R␣ B site. Costimulation, but not TcR/CD3 stimulation, appears supported by sustained down-regulation of both IB␣ and -␤ regulators. Furthermore, contrary to p65, c-Rel appears to display little affinity for p105, p100 and IB␣ regulators.

IL-2 and long-term T cell activation induce physical and functional interaction between STAT5 and ETS transcription factors in human T cells

“…For coimmunoprecipitation, 1 ml of whole cell extracts (10 7 cells) were incubated 1 h at 48C with 5 ml of antiserum. Immune complexes were precipitated with protein A-Sepharose (Pharmacia) as described (Kahn-Perles et al, 1997). For GST-pull down assays, 1 ml whole cell extracts (10 7 cells) were incubated 2 h at 48C with GST-recombinant proteins as shown in Figure 3 legend coupled to glutathione sepharose 4B resin (Pharmacia), followed by three washes with 0.1% NP-40 lysis bu er.…”

Nuclear Accumulation of cRel following C-Terminal phosphorylation by TBK1/IKKε

“…cRel is also phosphorylated and translocated to the nucleus following TCR stimulation by PMA/anti-CD28 (36), where nuclear cRel remains detectable for several days post-TCR stimulation (49). However, in contrast to RelA/p65, where mutation of Ser 536 to Ala is sufficient to abrogate IKK and TBK1-induced phosphorylation (24), cRel appears to have multiple sites for IKK -mediated phosphorylation.…”

A sustained activation of PI3K/NF-κB pathway is critical for the survival of chronic lymphocytic leukemia B cells

“…39 Nuclear proteins (2 mg) were incubated for 10 min in a final volume of 10 ml with 200 ng poly(dIdC) (Amersham Biosciences) before addition of 0.4 ng of 5 0 -end labelled ds-oligonucleotide kB, which contains an NF-kB binding site (5 0 CAA CGG CAG GGG AAT CTC CCT CTC CTT 3 0 ). 40 Binding reactions were allowed during 30 min at room temperature and electrophoresed in a 5% polyacrylamide gel in TBE (0.5 Â ). DNA-protein complexes were detected by X-ray film exposition.…”