Mapping Allosteric Sites on Protein Kinases Critical in MAPK Signaling

• Published in: The FASEB journal 2022-05, Vol.36 (S1), p.n/a
• Main Authors: Fera, Daniela, Lin, Sabrina, Naagaard, Daniel, Chen, Han, Finkelstein, Maxwell T., Glatz, Gwendolyn, Hayashi, Scout, Hotan, Quynn, Lau, Benjamin, LeBlanc, Julia, Li, Kevin, Parker Miller, Emma, Ye, Ming, Zaidi, Hussain
• Format: Article
• Language: English
• ISSN: 0892-6638; 1530-6860
• DOI: 10.1096/fasebj.2022.36.S1.R3001

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.