Regulation of protein phosphorylation within the MKK1–ERK2 complex by MP1 and the MP1•P14 heterodimer

MEK partner 1 (MP1) and P14 are small proteins that modulate the Raf-MKK1/2-ERK1/2 pathway. To examine the biochemical basis for their function a fluorescent form of MP1 was prepared by labeling Cys-74 with fluorescein. Using this protein it was shown that MP1 binds unactivated ERK1, ERK2 and a constitutively active form of MKK1 (MKK1G7B) with dissociation constants of 9.7 ± 1.6, 3.3 ± 0.6 and 2.2 ± 0.5 μM, respectively. MP1 inhibits the ability of activated ERK2 to phosphorylate the transcription factor Ets-1. Both MP1 and the MP1•P14 complex inhibit the ability of activated ERK2 to phosphorylate MKK1G7B, thus impeding feedback inhibition. In contrast, MP1 and the P14•MP1 complex enhance the ability of MKK1G7B to phosphorylate ERK2, when ERK2 is present at a low concentration, but not when it is present at a high concentration. Thus, MP1 and the MP1•P14 complex have the potential to differentially modulate activating and inhibiting signals in the Raf-MKK1/2-ERK1/2 pathway.