Hydrophobic as well as charged residues in both MEK1 and ERK2 are important for their proper docking

Docking between MEK1 and ERK2 is required for their stable interaction and efficient signal transmission. The MEK1 N terminus contains the ERK docking or D domain that consists of conserved hydrophobic and basic residues. We mutated the hydrophobic and basic residues individually and found that loss of either type reduced MEK1 phosphorylation of ERK2 in vitro and its abili ty to bind to ERK2 in vivo, Moreover, ERK2 was localized in both the cytopl asm and the nucleus when co-expressed with MEK1 that had mutations in eithe r the hydrophobic or the basic residues. We then identified two conserved h ydrophobic residues on ERK2 that play roles in docking with MEK1, Mutating these residues to alanine reduced the interaction of ERK2 with MEK1 in cell s. These mutations also reduced the phosphorylation of MEK1 by ERK2 but had little effect on phosphorylation of MBP by ERK2, Finally, we generated doc king site mutants in ERK2-MEK1 fusion proteins. Although the mutation of th e MEK1 D domain significantly reduced ERK2-MEK1 activity, mutations of the putatively complementary acidic residues and hydrophobic residues on ERK2 d id not change its activity. However, both types of mutations decreased the phosphorylation of Elk-l caused by ERK2-MEK1 fusion proteins. These finding s suggest complex interactions of MEK1 D domains with ERK2 that influence i ts activation and its effects on substrates.