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Our lab investigates molecular mechanisms that control dynamics in cell signaling. Currently, we are focusing on the function of internal motions in activation of ERK2, the role of the WRAMP protein network in rear-directed cell migration, and identification of signaling pathways underlying re-sensitization to drugs in melanoma.  All of our projects use the power of protein mass spectrometry to investigate changes in protein and cellular states. 

The role of protein dynamics in controlling the allosteric activation of ERK2

This project tackles the challenging question of how intramolecular protein dynamics are coupled to regulation of catalysis in enzymes. The MAP kinases, ERK1 and ERK2, promote cell proliferation, survival, migration and invasion, and are emerging therapeutic targets for treating cancer.  We have discovered that dual phosphorylation and activation of ERK2 leads to changes in protein dynamics, resulting in global motions of the activated state. The motions can be modeled as a slow, reversible equilibrium between two conformational states, which shift the kinase between locked (“L”) to released (“R”) conformations to allow productive ATP binding in the active enzyme.

To study this problem, we use a combination of hydrogen-deuterium exchange mass spectrometry (HX-MS), NMR multiple quantum Carr-Purcell-Meiboom-Gill relaxation dispersion measurements (CPMG-NMR), and X-ray structural analyses to dissect the relationship between protein structure and dynamics, and how these contribute to the regulation of catalysis. A recent discovery is that two tight-binding inhibitors of ERK2, Vertex-11e and SCH772984, affect the dynamics of the kinase and enable conformational selection between different states. We are investigating how conformational selection affects the inhibitory properties of different molecules targeting ERKs.

Control of directional cell movement by the WRAMP structure

The ability of cells to migrate directionally is critical for organismal development, tissue repair, and immunity, as well as cancer cell invasion and metastasis. We have discovered a protein network named the “Wnt5a-receptor-actomyosin-polarity (WRAMP) structure” which polarizes to the rear of mammalian cells, followed by an immediate retraction of the rear membrane.  Our evidence shows that WRAMP structures confer cells with faster speed and longer persistence of directional movement.  WRAMP structures also form dynamically, and when they change their location, cells change direction, suggesting a role for WRAMP structures in determining the direction of migration.  These findings highlight the importance of cell signaling at the rear of cells for directed cell migration and suggesting a role for the WRAMP structure in determining the direction of migration by establishing the location of the cell rear.

We are currently investigating components within WRAMP structures, their molecular organization, and their order of assembly.  WRAMP structure components so far include cell adhesion molecules (MCAM, ICAM and CD44), F-actin and myosin-II, actin-binding proteins, and Wnt signaling molecules. In addition, late endosomes and cortical endoplasmic reticulum endomembranes are found at WRAMP structures suggesting the involvement of organelle trafficking mechanisms in rear-directed polarity. WRAMP structure formation is followed by localized Rho/ROCK activation and a transient spike of cytosolic Ca²⁺, followed by membrane retraction.  Thus, WRAMP structures elicit a rear-polarized elevation of Rho and second messenger signaling, which we think provides the driving force for rear membrane movements.  Approaches include superresolution imaging in live and fixed-cells, proximity-based protein labeling proteomics (BioID, APEX), and organelle proteomics and correlation profiling, to identify new protein components at the WRAMP structure, determine their molecular interactions, and elucidate their dynamics of assembly.