Epi-allelic Erk1 and Erk2 knockdown series for quantitative analysis of T cell Erk regulation and IL-2 production

Abstract

Erk activation is often used as a downstream pathway indicator of TCR signaling, generally in terms of both Erk1 and Erk2 isoforms measured together. In order to investigate potential distinctions between Erk1 and Erk2 regulation and effects downstream of TCR ligation, we generated a series of stable and independent Erk1 and Erk2 shRNA knockdown lines in the 1B6 T cell hybridoma. We observed no compensatory effect by opposite isoform upregulation, and found similar fractions of total phosphorylated Erk1/2 across this epi-allelic series in response to both anti-CD3 and peptide-MHC stimulation of TCR. Moreover, a previous prediction of an isoform-independent linear relationship between Erk activation and IL-2 production was confirmed. The effect of the shRNAmediated knockdowns in reducing IL-2 production was observed to be stronger than that arising from pharmacological MEK inhibition at comparable degrees of ERK1/2 phosphorylation levels.

Vitamin D receptor upregulation in alloreactive human T cells

“…We examined the association between VDR upregulation and ERK1/2 given the known importance of MAPK signaling in human T cell activation and IL-2 production [32]. Our finding that phosphorylation of ERK1/2 precedes expression of VDR strictly on p-ERK1/2+ cells suggests a possible role for the ERK1/2 pathway in VDR upregulation.…”

“…Prior studies have demonstrated that GVHD-inducing alloreactivity is mediated primarily by naïve and possibly early memory T cells [29,30] and also may depend on ERK1/2 activation [31], which is important for IL-2 production [32]. Therefore, we developed an approach to assess VDR and p-ERK1/2 simultaneously, and examined the maturation stage dependence of VDR upregulation.…”

MPYS, a Novel Membrane Tetraspanner, Is Associated with Major Histocompatibility Complex Class II and Mediates Transduction of Apoptotic Signals

“…The forward and reverse primers (1 l each of 1 g/l) (see Table S2 in the supplemental material) were annealed (28), and the annealed oligonucleotides were ligated into pLL3.7 through HpaI and XhoI sites. An shRNA that targets the luciferase gene was used as a control (31). Lentiviruses were generated as previously described (28).…”

Integrated network analyses for functional genomic studies in cancer

“…More specifically, siRNA offers the advantage of isoform specificity and enable fine-tuning of individual isoform expression and activity. For an investigation of T-cell Erk regulation, researchers used epi-allelic series with siRNAs against ERK1 and ERK2 to identify the role of these kinases on downstream IL-2 production [17]. The epi-allelic series again showed a correlation between siRNA targeting efficiency and phenotype.…”