Introduction
MAP kinase-activated protein kinase 2 (MAPKAP-kinase 2) was originally identified as a substrate
for the p42/p44 MAPKs in vitro (1). However, recent data indicate that in intact cells, the upstream kinase
that regulates MAPKAP-kinase 2 is p38 MAPK (2-5). Treatment of cells with endotoxin, interleukin-1,
tumor necrosis factor, or various stress stimuli activate p38 MAPK and MAPKAP-kinase 2 (2-5). Mice
deficient in MAPKAP-kinase 2 showed a reduction in bacterial lipopolysaccharide-induced biosynthesis
of tumor necrosis factor (TNF)-, interferon-, interleukin (IL)-1, IL-6, and nitric oxide (6), suggesting a
critical role of MAPKAP-kinase 2 in inflammatory cytokine production. In human neutrophils
MAPKAP-kinase 2 is also activated by the chemotactic factor fMet-Leu-Phe and phorbol 12-myristate
13-acetate (7). The function of MAPKAP-kinase 2 is not known, but its upstream kinase p38 MAPK has
been proposed to be involved in the biosynthesis of inflammatory cytokines (8), apoptosis (9) and platelet
aggregation (10). MAPKAP-kinase 2 contains a C-terminal autoinhibitory domain (10-13) and is activated
by phosphorylation at multiple sites (12, 13).
Recently it was reported that the major substrate for MAPKAP-kinase 2 in human neutrophils is not
HSP27 but is a protein termed p60 (14), which was identified as LSP1 (Leukocyte Specific Protein 1), a
339-amino acid cytoskeletal protein, the expression of which is restricted to neutrophils, lymphocytes,
and macrophages.
Measurement of MAPKAP-kinase 2 activity
The protocol generally regarded as most sensitive for the quantitative measurement of
MAPKAP-kinase 2 activity involves incubation of the MAPKAP-kinase 2 sample with substrate (either
a natural or synthetic polypeptide such as Long S6 Kinase substrate peptide), in the presence of Mg2+and
32P-labeled ATP. The reaction is terminated by "spotting" a sample onto a filter paper disc, followed by
immersion in acid to precipitate the radiolabeled product. The filter papers are then washed extensively
to remove unincorporated radiolabel and the radioactivity is counted. While sensitive, this method is
labor-intensive, generates hazardous radioactive waste and depends on a radioisotope with a short
half-life. It is particularly unsuitable when kinase assays are only performed on an infrequent basis. The
MAPKAP-kinase 2 assay/Inhibitor Screening Kit uses a peroxidase coupled anti-phospho-LSP1
serine204 monoclonal antibody as a reporter molecule in a 96-well ELISA format. This assay provides a
non-isotopic, sensitive, and specific method to detect MAPKAP-kinase 2 activity.