ERK2 phosphorylation site mutants

The MAP kinase ERK2 is activated by phosphorylation on two sites, Y185 and T183, that lie in the phosphorylation lip at the mouth of the protein kinase active site. In the low activity enzyme, Y185 is buried, while T183 is on the surface. Through a crystallographic analysis of phoshorylation site mutants of ERK2 we have identified nineteen residues that are likely to be involved in the conformational changes associated with activation. When T183 is replaced by glutamate, few conformational changes are observed. By contrast, when Y185 is substituted by glutamate, the structure of the phosphorylation lip and an ajacent loop is lost. A conservative substitution, Y185F, also induces relatively large conformational changes. The flexibility of the lip observed in the mutants suggests that the entire lip can refold to conform to the catalytic site of the activating enzyme, and refold on phosphorylation to form the active structure. The ability of the lip to change its structure has implications for the mechanisms of regulation of other protein kinases as well. This data shows that the integrity of lip in the inactive conformation is dependent specifically on Y185. High resolution refinement of unphosphorylated wild-type ERK2 reveals a putative binding site on the surface of ERK2 for the phosphorylated form of Y185. Thus we propose that, in addition to its role in tethering the phosphorylation lip in the inactive conformation of ERK2, Y185 also has a role in forming the active structure.

Activity of MAP kinase ERK2 is controlled by a flexible surface loop.

Jiandong Zhang, Faming Zhang, Douglas Ebert, Melanie H Cobb and Elizabeth J Goldsmith.
Structure, 3:299-307(1995)