The impact of tyrosine kinase 2 (Tyk2) on the proteome of murine macrophages and their response to lipopolysaccharide (LPS)

Abstract

Tyrosine kinase 2 (Tyk2) belongs to the Janus kinase (Jak) family and is involved in signalling via a number of cytokines. Tyk2-deficient mice are highly resistant to lipopolysaccharide (LPS)-induced endotoxin shock. Macrophages are key players in the pathogenesis of endotoxin shock and, accordingly, defects in the LPS responses of Tyk2(-/-) macrophages have been reported. In the present study, the molecular role of Tyk2 is investigated in more detail using a proteomics approach. 2-D DIGE was applied to compare protein patterns from wild-type and Tyk2(-/-) macrophages and revealed significant differences in protein expression patterns between the genotypes before and after LPS treatment. Twenty-one proteins deriving from 25 differentially expressed spots were identified by MALDI/ESI MS. Among them, we show for N-myc interactor that its mRNA transcription/stability is positively influenced by Tyk2. In contrast, LPS-induced expression of plasminogen activator 2 protein but not mRNA is strongly enhanced in the absence of Tyk2. Our data furthermore suggest an influence of Tyk2 on the subcellular distribution of elongation factor 2 and on LPS-mediated changes in the peroxiredoxin 1 spot pattern. Thus, our results imply regulatory roles of Tyk2 at multiple levels and establish novel connections between Tyk2 and several cellular proteins.

Transcriptome analysis reveals a major impact of JAK protein tyrosine kinase 2 (Tyk2) on the expression of interferon-responsive and metabolic genes

“…Our findings are also consistent with our previous observation that Tyk2 modulates metabolic proteins in BMDMs before and after LPS treatment [29]. However, it remains possible that isolated mouse macrophages display somewhat abnormal metabolic behavior.…”

A comparative proteome analysis links tyrosine kinase 2 (Tyk2) to the regulation of cellular glucose and lipid metabolism in response to poly(I:C)

“…Following cultivation for 6–7 days, cells were incubated with 50 μg/ml poly(I:C) or 500 units/ml IFNβ for the times indicated. If not stated otherwise, whole cell lysates were prepared as previously described [12] and used for 2D-DIGE and Western blot analysis. Alternatively, RIPA buffer (20 mM Tris–HCl pH 7.4, 150 mM NaCl, 1% Triton-X-100, 0.5% Na-deoxycholate; 0.1% SDS and protease inhibitors as described [12]) was used.…”

“…If not stated otherwise, whole cell lysates were prepared as previously described [12] and used for 2D-DIGE and Western blot analysis. Alternatively, RIPA buffer (20 mM Tris–HCl pH 7.4, 150 mM NaCl, 1% Triton-X-100, 0.5% Na-deoxycholate; 0.1% SDS and protease inhibitors as described [12]) was used. Protein concentration was measured by the Coomassie G-250 (dye reagent, Bio-Rad laboratories, Hercules, CA, USA) protein binding assay [15].…”

“…DIGE labeling, 2-DE separation and evaluation were performed as previously described [12]. Briefly, samples were minimally labeled with CyDye DIGE™ fluorescent dyes (GE Healthcare Life Sciences, Munich, Germany).…”

“…In a previous study, we used a proteomic approach to investigate the molecular role of Tyk2 in macrophages in response to LPS, a structural membrane component of Gram-negative bacteria recognized by TLR4 [12]. The absence of Tyk2 showed complex consequences on the macrophage proteome and implied regulatory roles of Tyk2 at the mRNA and post-transcriptional level before and after treatment with LPS.…”