Figure 2.

Expression of dominant-negative or activated MEK5 in T cells. (A) Representation of the retrovirus construct for expression of MEK5(A) or MEK5(D). This construct drives co-expression of GFP under IRES, allowing identification of cells that are retrovirally transduced. (B) The effects of MEK5(A) and MEK5(D) overexpression on ERK5 activation responses. Total lysates from empty, MEK5(A), or MEK5(D) retrovirus-transduced DO11.10 hybridoma cells were analysed by immunoblotting for phospho-ERK5 (upper) or total ERK5 (lower) at indicated time points following stimulation with ionomycin at 0.5 μM and PMA at 2.5 ng/ml. MEK5(A) inhibits, whereas MEK5(D) enhances, ERK5 activation. (C) The effects of MEK5(A) and MEK5(D) on ERK5 target gene expression in T cells. Retrovirally transduced DO11.10 hybridoma cells were stimulated for 6 h with ionomycin and PMA and were analysed by quantitative RT–PCR for expression of known ERK5 target genes (nur77 and lklf), apoptotic gene (bim), a Nur77-related family member (nor-1) or ERK1/2 target gene (egr-1). Relative values were calculated by normalization against corresponding HPRT values and expressed as the mean. Error bars indicate standard deviation of triplicate samples. Data from a representative experimental set are shown. (D) Responses of nur77, nor-1, bim, lklf, and egr-1 genes to activated ERK5 in primary thymocytes. Thymocytes were transiently transfected with pCI (empty) or pCI-MEK5(D), stimulated in vitro with anti-CD3 and anti-CD28 antibodies for 4 h and analysed by quantitative RT–PCR. These values were normalized against HPRT. The average values of triplicate samples are shown, with the error bars indicating standard deviation. Results representative of three independent experiments are shown.