MAP KINASES FROM BOVINE BRAIN - PURIFICATION AND CHARACTERIZATION

We have purified 42- and 44-kilodalton (kDa) isoforms of the mitogen-a ctivated protein (MAP) kinase family from bovine brain. The kinases we re assayed with myelin basic protein as the substrate and detected by anti-sea star p44(mpk) antibody. Purification was achieved using pheny l-Sepharose, polylysine-agarose, hydroxylapatite, and Mono-Q column ch romatography. Both myelin basic protein and smooth muscle caldesmon, b ut not histone H1, served as good substrates. Based on chromatographic behaviors and specific activities toward myelin basic protein, it is likely that the 42-kDa brain isoform is similar to that of brain tau k inase. The 44-kDa enzyme, however, is a novel brain MAP kinase isoform not reported previously. Although it has been demonstrated that p44(m pk) can be activated in vitro through phosphorylation by the tyrosine kinase p56(lck), neither of the brain kinases were significantly stimu lated by the tyrosine kinases p56(lck), p56(lyn), or p59(fyn). However , based on antibody cross-reactivity, a MAP kinase kinase is present i n the crude brain extract. Both brain MAP kinases were capable of auto phosphorylation which occurred, at least in part, on tyrosine residues . However, only the 44-kDa isoform showed a significant degree of coin cident activation.