Mitogen-activated
protein (MAP)
kinases are a family of
serine/threonine protein kinases that play an important role in a myriad of cellular processes, including cell proliferation, differentiation, and apoptosis. Abnormal activation of MAP
kinases has been shown to participate in a variety of human diseases which include
cancer,
septic shock,
rheumatoid arthritis, diabetes, and
cardiovascular diseases. Active MAP
kinase enzymes are not only valuable for basic biomedical research but are also critical for the development of pharmacological inhibitors as therapeutic drugs in the treatment of relevant human diseases. MAP
kinases produced in a bacterial system are poorly active due to a lack of proper phosphorylation at their characteristic
threonine and
tyrosine residues. To overcome these limitations, we have developed a mammalian expression system for high level expression and one-step purification of enzymatically MAP
kinases. We cloned JNK1, p38, and p38-regulated MAP
kinase-activated
protein kinase-2 into the mammalian expression vector pEBG, and expressed these
protein kinases as
glutathione S-transferase fusion
proteins in human embryonic kidney 293T cells through transient transfection. The
protein kinases were activated in vivo through treating the transfected cells with
sodium arsenite and affinity-purified using
glutathione-
Sepharose beads. The enzymatic activities of these
protein kinases were demonstrated by Western blot analysis and in vitro
kinase assays. Our results indicate that this system is an extremely powerful tool for generating valuable
reagents, and could be very valuable for proteomic studies.