Cryo-EM structures of phosphorylated MEK1 bound to ERK1 reveal that MEK1 interacts with ERK1 through three specific interfaces and functions as a multifunctional enzyme that not only catalyzes canonical ATP-dependent phosphorylation of ERK1 at Y204 and T202, but also exhibits phosphatase activity to dephosphorylate Y204 and catalyzes an unprecedented relay mechanism in which the phosphate group is transferred from Y204 to T202, establishing a dual-pathway catalytic cycle driven by nucleotide-dependent changes in binding affinity.

The RAS-RAF-MEK-ERK cascade regulates critical cellular processes such as cell proliferation and differentiation, with its dysregulation driving over 30% of human cancers. However, the active state of MEK and molecular mechanisms of ERK phosphorylation remain unclear. Here, we report the cryo-EM structures of phosphorylated MEK1 (pMEK1) bound to unphosphorylated and monophosphorylated ERK1. These structures reveal that pMEK1 interacts with ERK1 via three interfaces: first between MEK1 N-terminal peptide and ERK1 D-site recruitment site (DRS), second between MEK1 C-lobe and ERK1 F-site recruitment site (FRS), and third between MEK1 activation loop and ERK1 activation loop. Notably, we identify an unexpected phosphorylation mechanism: pMEK1 catalyzes ERK1 T202 phosphorylation through phosphate transfer from ERK1 Y204. In addition, pMEK1 exhibits phosphatase activity, catalyzing the dephosphorylation of ERK1 Y204. These findings allow us to delineate the catalytic cycle of MEK-catalyzed ERK phosphorylation and provide insights for targeting this oncogenic pathway.