(489.11) Biochemical analysis of human ERK2 mutants reveals important residues in MEK1-ERK2 binding and phosphorylation

Poster Board Number: A81

Ming Ye (Swarthmore College), Quynn Hotan (Swarthmore College), Gwendolyn Glatz (Swarthmore College), Maxwell Finkelstein (Swarthmore College), Scout Hayashi (Swarthmore College), Emma Miller (Swarthmore College), Benjamin Lau (Swarthmore College), Kevin Li (Swarthmore College), Han Chen (Swarthmore College), Julia LeBlanc (Swarthmore College), Hussain Zaidi (Swarthmore College), Daniela Fera (Swarthmore College)

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.

Support or Funding Information

Funding: This work was supported by The Pittsburgh Foundation (UN2020-114825 awarded to DF).