T-cell activation requires signals from both the T-cell receptor (TcR) and other co-stimulatory molecules such as CD28. TcR- and CD28-mediated signals are integrated during T-cell activation resulting in the expression of cytokine genes such as interleukin-2 (IL-2). An enhancer element (CD28RE) of the IL-2 gene specifically responsive to CD28 signals has been previously identified and characterized. This response element and an adjacent Activated Protein-1 (nuclear factor-interleukin-2B) site together (RE/AP1) were shown to complex with c-rel, AP-1 and other factors. However, details of the signal transduction pathways leading from CD28 to the composite response element remain poorly understood. We present data showing that overexpression of the serine threonine kinase, mitogen-activated protein kinase/extracellular-signal-regulated kinase kinase kinase-1 (MEKK1), but not nuclear factor-κB inducing kinase, or MAP kinase/ERK kinase-1 (MEK1), can significantly increase the level of CD28RE/AP1-driven luciferase (Luc) reporter gene expression in Jurkat E6-1 cells. A MEKK1 dominant negative mutant blocked such activation induced by stimulation with Raji B cells and the superantigen staphylococcus enterotoxin E (SEE), as well as via CD3/CD28. Mutations in either site of the RE/AP1 element abolished MEKK1-induced Luc expression. Calcineurin inhibitors, CsA and FK520, or inhibitors of p38 kinase (SB 203580), or MEK1 (PD 098059), did not affect MEKK1-induced reporter activation. These results directly implicate MEKK1 in the CD28 signalling pathway that activates the CD28 response element. Co-expression of the lymphocyte-oriented kinase (LOK) kinase attenuated Raji/SEE-induced IL-2 production in Jurkat cells, as well as MEKK1 and Raji/SEE-induced reporter gene activation. These data suggest that MEKK1 and LOK may have opposing roles in regulating the CD28RE/AP1 element.
Abbreviations used: AP-1, Activated Protein-1; CD28RE, CD28 responsive element; Cot, cancer Osaka thyroid; EGFP, enhanced green fluorescent protein; ERK, extracellular signal-regulated kinase; Grb2, growth-factor-receptor-bound protein 2; IKK, IκB kinase; IL-2, interleukin-2; ITK, inducible T-cell kinase; JNK, c-Jun N-terminal kinase; JNKK, JNK kinase; LOK, lymphocyte-oriented kinase; LOKKinGFP, LOK kinase domain fused to GFP; Luc, luciferase; MAP, mitogen-activated protein; MAPK, mitogen-activated protein kinase; MEK1, MAP kinase/ERK kinase-1; MEKK1, MEK kinase-1; MKK, MAPK kinase; NFAT, nuclear factor of activated T-cells; NF-κB, nuclear factor κB; NF-IL-2B, nuclear factor-interleukin-2B; NIK, NF-κB inducing kinase; PAK, p21-activated kinase; PI3K, phosphatidylinositol-3-phosphate kinase; P/I, PMA/ionomycin; RE/AP, responsive element/AP; SEE, staphylococcus enterotoxin E; SEK, stress-activated protein kinase/ERK kinase; TcR, T-cell receptor.