The Ras‐Raf‐MEK‐ERK (MAPK) pathway is a signal transduction cascade used to regulate cellular processes including cell cycle progression and proliferation. Aberrant activation of this pathway is implicated in cancer development, and treatment with Raf, MEK and ERK inhibitors often leads to adaptive resistance. Combination therapies have been shown to offer benefits; however, the MEK‐ERK interface remains poorly understood, hindering structure‐guided approaches to the design of potent MAPK pathway inhibitors targeting this complex. To identify important residues for the MEK1‐ERK2 interaction, we performed site‐directed mutagenesis on ERK2. We used circular dichroism to assess the secondary structure of the ERK2 mutants. We also used biolayer interferometry binding experiments coupled with phosphorylation assays to evaluate the impact of the mutated residues on the formation of the MEK1‐ERK2 complex and activity. Circular dichroism showed no differences in secondary structure for any of our ERK2 mutants. Of all the mutations generated, the L234D mutation in ERK abrogated binding and phosphorylation by MEK1 the most. Other mutants showed some reductions in binding or activity but require further analysis. Of note, L234 is located on an ERK2 α‐helix adjacent to the phosphorylation lip, consistent with MEK1 binding this face during phosphorylation. Our results suggest that this α‐helix may play critical roles in the MEK1‐ERK2 complex. Studying the impact of additional mutations in this and additional regions will develop our understanding of the MEK‐ERK interface and inform the design of allosteric inhibitors that can modulate MEK‐ERK complex formation.

The mitogen‐activated protein kinase (MAPK) pathway plays a critical role in controlling cell cycle progression and cell proliferation. This Ras‐Raf‐MEK‐ERK pathway is constitutively activated in over 80% of melanomas, as well as in some pancreatic, colon, lung, ovarian, and kidney cancers. Anticancer drugs targeting proteins earlier in the pathway, such as B‐Raf and MEK, often lose efficacy due to the development of resistance. Thus, drugs that target allosteric sites hold promise if used in combination with ATP‐competitive inhibitors that have already been developed. We have been working on identifying allosteric sites on key kinases of this pathway that could be used for future drug discovery efforts. We previously found that the alpha‐G helix of B‐Raf is critical for its interaction with MEK and that mutations of several residues along this helix could completely abrogate binding and downstream phosphorylation activity in vitro. With this information, we further analyzed the crystal structure of the B‐Raf‐MEK complex to generate mutations in MEK to determine if the same region of MEK that contacts the B‐Raf alpha‐G helix is important for binding ERK. Additionally, since no complex structure of the MEK‐ERK complex was available, we generated mutations in different regions of the C‐lobe of ERK as a starting point to identify a region of contact. We tested the effects of our mutations on binding using pull downs and biolayer interferometry. We also assessed phosphorylation levels of ERK in vitro. Our MEK mutants displayed no difference in binding to B‐Raf, but did have altered binding to ERK. This suggests that the modes of MEK binding to B‐Raf versus ERK differ. Moreover, our mapping of the ERK interface has led to the identification of a residue on the helix spanning residues 232‐245 that, when mutated, significantly reduces both binding to MEK and phosphorylation of ERK. Additional work is needed to determine if our mutation affects the conformation of a nearby alpha helix of ERK and as a result affects MEK binding or if it is due to a direct effect. Nevertheless, these results demonstrate that MEK‐ERK binding and downstream activity can be altered by targeting sites outside their catalytic region.