(489.14) Investigating the Interaction Interface between MEK and ERK Kinase

Poster Board Number: A84

Sabrina Lin (Swarthmore College), Mats Naagaard (Swarthmore College), Tammy Pham (Swarthmore College), Daniela Fera (Swarthmore College)

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.

Support or Funding Information

This work was supported by The Pittsburgh Foundation (UN2020-114825 awarded to Daniela Fera).