To ensure that MKK7 actually directly phosphorylates ERK1/2, we produced recombinant ERK2 in bacteria, and purified naïve and phosphorylated MKK7 by immunoprecipitation from human neurons that overexpressed Flag-tagged MKK7 and that had been treated with control medium or ApoE3 (Fig. 3C). We then incubated recombinant ERK2 with immunoprecipitated naïve or ApoE3-activated MKK7 in the absence and presence of the MEK inhibitor U0126, and measured ERK2 phosphorylation. We observed that MKK7 directly phosphorylated ERK2; ERK2 phosphorylation by MKK7 was enhanced by prior ApoE3-dependent activation of MKK7, and blocked by U0126 (Fig. 3C). These data confirm that ApoE activates a non-canonical MAP-kinase signal transduction pathway consisting of a DLK→MKK7→ERK1/2 phosphorylation cascade (Fig. 3A).
