MAP KINASES AND THEIR ACTIVATING ENZYMES. Elizabeth J. Goldsmith, Department of Biochemistry, UT Southwestern Medical Center at Dallas, Dallas, TX 75235- 9050

The ras- activated protein kinase cascades are transducers of extracellular signals and differentially participate in the signaling of transformation, proliferation, and differentiation. Each cascade is a module consisting of three enzymes, a MAP kinase and two upstream activating enzymes. The MAP kineses are tightly regulated by dual; phosphorylation events, one on a tyrosine and one a threonine residue. We have determined the structure of both the low activity unphosphorylated form and the high activity doubly phosphorylated form. The results show that regulation of activity is conferred by the conformation of the phosphorylation lip near the active site and the relative orientation of the two domains of the molecule. The phosphorylation site tyrosine is found buried in the low activity enzyme structure. Apparently conformational changes take place as the MAP kinase is bound to its activating enzyme, MEK. Analysis of the structures of MAP kinase mutants shows that the phosphorylation lip becomes disordered when negative charges are introduced in place of the phosphorylation site residues. Apparently, this region is relatively unstable, thus allowing small binding energies, derived from interaction with the activating enzyme, to dislodge the lip for phosphorylation.

We also have undertaken to determine the structures of several other protein kineses belonging to MAP kinase pathways, and have determined the structure of the MAP kinase homolog activated in response to cytokines, P38.