MAPKAP kinase 2 (MK2) is one of several kinases that are regulated through direct phosphorylation by p38 MAP kinase. By introducing a targeted mutation into the mouse MK2 gene, we have determined the physiological function of MK2 in vivo. Mice that lack MK2 show increased stress resistance and survive LPS-induced endotoxic shock. This is due to a reduction of approximately 90% in the production of tumor necrosis factor-alpha (TNF-alpha) and not to a change in signalling from the TNF receptor. The level and stability of TNF-alpha mRNA is not reduced and TNF-alpha secretion is not affected. We conclude that MK2 is an essential component in the inflammatory response which regulates biosynthesis of TNF-alpha at a post-transcriptional level.

We demonstrate that lipopolysaccharide-induced tumor necrosis factor (TNF) biosynthesis becomes independent of MAPKAP kinase 2 (MK2) when the AU-rich element (ARE) of the TNF gene is deleted. In spleen cells and macrophages where TNF biosynthesis is restored as a result of this deletion, interleukin (IL)-6 biosynthesis is still dependent on MK2. In MK2-deficient macrophages the half-life of IL-6 mRNA is reduced more than 10-fold, whereas the half-life of TNF mRNA is only weakly decreased. It is shown that the stability of a reporter mRNA carrying the AU-rich 3-untranslated region (3-UTR) of IL-6 is increased by MK2. The data provide in vivo evidence that the AU-rich 3-UTRs of TNF and IL-6 are downstream to MK2 signaling and make MK2 an essential component of mechanisms that regulate biosynthesis of IL-6 at the levels of mRNA stability, and of TNF mainly through TNF-ARE-dependent translational control.

We describe a novel parallel method for the patterning of proteins with nanoscale resolution. Combining nanoimprint lithography (NIL) and molecular assembly patterning by lift-off (MAPL), we produced streptavidin patterns with feature sizes in the order of 100 nm. A stamp is imprinted into a heated PMMA film followed by a dry etching step that converts the topography into a PMMA/Nb 2 O 5 contrast. A biotin functionalized copolymer, poly(L-lysine)-graft-poly(ethylene glycol)-biotin (PLL-g-PEG/PEG-biotin), spontaneously adsorbs on the oxide surfaces. After PMMA lift-off, the background is backfilled with protein-resistant PLL-g-PEG. We show that streptavidin selectively adsorbs on the biotin areas and thus can be used as a universal platform for immobilization of biotin-tagged molecules. This novel process is a parallel patterning method that is fast, reproducible, and economic. The PEG-copolymer can be functionalized with a variety of bioactive groups and thus allows a great flexibility in terms of surface chemistry.