Proteomics and Signal Transduction
Our goal is to discover new mechanisms underlying the regulation and function of cell signaling. A major aim is to develop and apply new methods for protein profiling by mass spectrometry with biochemical and cellular approaches to investigate cellular responses to signaling pathways. A second aim is to examine internal motions in protein kinases, demonstrating coupling between protein dynamics and catalytic function.
We investigate cancer progression by investigating signaling pathways that are activated in melanoma and influence cancer progression and cell behavior (Fig. 1). Current studies include projects which focus on signaling through Wnt5A and B-Raf. Past work has examined signaling through RhoA GTPases, whose activation correlates with metastatic melanoma.
Wnt5A signaling
Wnt5A, which normally controls embryonic body axis formation, is elevated in high-grade melanomas and promotes cell invasion. We discovered a novel protein-organelle complex, which directs lifting and retraction of tail-end plasma membrane during directional cell movement. The complex, named the “Wnt5a receptor-actin-myosin-polarity (WRAMP) structure" involves the MCAM/CD146/MUC18 cell adhesion receptor, which polarizes intracellularly, dynamically recruiting actin and myosin-II to the cell posterior where it triggers membrane retraction and moves cells forward (Fig. 1). Whereas cell polarity studies often focus on events at the cell leading edge, the WRAMP structure shows that signaling at the rear is equally important for directional cell movement.
We identified components of the WRAMP structure by organelle proteomics, and discovered subunits of vesicle coatomer-I (COP-I), which are localized to and required for WRAMP structure formation. We found that endoplasmic reticulum (ER) is recruited to the WRAMP structure and cortical plasma membrane, followed by cytosolic calcium mobilization. Thus, recruitment of cortical ER produces a rear-directed calcium gradient, allowing actomyosin contraction and focal adhesion disassembly, events needed for tail-end membrane lifting and retraction. This reveals a new concept for cell polarity, in which cell movement is controlled by polarized endosomal trafficking and cortical ER recruitment.
B-Raf signaling
B-Raf is mutated in ~50% of melanomas, enhancing cell transformation, invasion and metastasis. We developed a large-scale strategy for phosphoprotein profiling, using negative precursor ion mass spectrometry, first described by Steven Carr (Broad Institute, MIT) and Roland Annan (GlaxoSmithKline), to selectively detect and quantify phosphopeptides based on their fragmentation signature following release of PO3– (Fig. 2). The method bypasses the need for metabolic cell labeling with stable isotope-labelled amino acids, and for phosphopeptide enrichment using affinity resins, which do not recognize all peptide chemistries.
Phosphoprotein targets that we identified included FAM129B/MINERVA, a protein with unknown function. We demonstrated that FAM129B functions in promoting 3D-invasion via phosphorylation by MAPK/ERK, localizes to the WRAMP structure and is required for its assembly. Current studies are aimed at investigating mechanisms important for cancer therapies targeting B-Raf/MKK/ERK signaling, comparing PLX4032/vemurafenib, an inhibitor of B-Raf-V600E which is approved for treating advanced melanomas, and the MKK1/2 inhibitor, AZD6244/selumetinib, which elicits poor responses in patients despite impressive pre-clinical success. We are keen to learn what determines the efficacy of different kinase inhibitors which target the B-Raf/MKK/ERK pathway.
Proteomics Technologies
Many studies in our lab implement large-scale proteomics by multidimensional LC-MS/MS, where proteins in complex mixtures are proteolyzed in solution, and peptides are separated by multi-dimensional liquid chromatography and sequenced by MS/MS. Currently, we are able to identify 8,000 proteins in a single 2D-LC-MS/MS run. Limitations remain in validating peptide sequences, inferring protein identity from peptide information, quantifying protein abundances, and phosphorylation site mapping. We are developing new experimental and computational strategies to address these problems. A key goal is to improve the accuracy and sensitivity of peptide assignments, using kinetic modeling of gas phase fragmentation to evaluate chemical plausibility of MS/MS spectra, and a "peptide-centric" database to reduce ambiguities in protein identification.
Protein Kinase Dynamics
We use hydrogen-exchange mass spectrometry (HX-MS) to reveal internal protein motions in kinases, where exchange predominantly occurs through low energy fluctuations in structure, allowing transient solvent exposure of backbone amides. Our goal is to address how protein dynamics regulates catalytic activity in protein kinases and other enzymes. The MAP kinase, ERK2, provides an ideal system, because its dynamics are clearly linked to activity, and active vs inactive forms of wild-type enzyme can be directly compared.
Our studies of ERK2 have revealed that kinase activation enhances HX at the linker between N- and C-terminal domains, which we ascribe to increased backbone flexibility. We showed that active (2P) vs inactive (0P) forms of ERK2 differ in their HX protection patterns upon nucleotide binding, reflecting closed vs open conformations, respectively (Fig. 3). Thus, ERK2 is constrained from domain closure before phosphorylation, and part of the activation mechanism involves releasing these constraints to form a competent catalytic site.
Current work integrates HX-MS and NMR studies of ERK2 with site-directed mutagenesis to identify relevant residues linking enzyme dynamics and catalytic turnover. We envision that motions of some residues influence catalysis, while others regulate linker flexibility through side-chain and backbone interconnectivities linked to the phosphorylation sites. A fascinating finding is that ERK1, which is closely related in sequence and structure to ERK2, differs significantly in its regulation of domain closure by phosphorylation. These differences are being exploited to identify mechanisms conveying motional information from the phosphorylation site to the linker region in ERK2. Eventually, targeting mechanisms which control protein dynamics may be exploitable for the design of inhibitors with selectivities that are difficult to achieve with ATP analogues.
