The mitogen‐activated protein kinase (MAPK) pathway is pertinent to various cellular processes such as cell proliferation, cell division, and cell death. Dysregulation of the MAPK pathway can therefore lead to numerous illnesses. Most notably, the dysregulation of proteins in this pathway is a significant cause of melanomas and other cancers. Due to this oncogenic potential, various drugs and inhibitors exist to regulate MAPK overexpression. As drug resistance and paradoxical activation are perpetual challenges, additional regulatory targets are needed to effectively suppress the effects of MAPK oncogenic mutations, calling for further investigation into downstream interactions such as that between MEK and ERK kinase. By identifying and characterizing the binding interface between MEK and ERK, we can identify additional drug targets that can modulate the MAPK pathway. To help map the MEK‐ERK interface, we first performed computational docking experiments to find possible interaction regions between MEK and ERK. In several trials, we found that certain MEK alpha helices interacted with ERK. We then proceeded to identify the corresponding binding partners on ERK. We introduced mutations in the most promising regions on ERK and expressed the mutant proteins using a bacterial expression system. Using biolayer interferometry and co‐elution assays, we compared the binding affinities of our mutants to that of the corresponding wild‐type. Our studies showed that introducing the K231E and K114E mutations in ERK altered the binding kinetics between MEK and ERK. Our co‐elution studies showed that a stable complex does not occur, suggesting that the interaction between MEK and ERK is either weak or transient and may need another molecule to stabilize the complex. Cross‐linking experiments are in progress, and so far, we identified 1% glutaraldehyde incubated for 5 minutes as sufficient for covalent linkage of MEK and ERK. These findings will help towards obtaining a 3D MEK‐ERK complex structure to better understand signaling in this pathway.

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.