The ERK mitogen-activated protein kinase signaling network: the final frontier in RAS signal transduction

The RAF–MEK–ERK mitogen-activated protein kinase (MAPK) cascade is aberrantly activated in a diverse set of human cancers and the RASopathy group of genetic developmental disorders. This protein kinase cascade is one of the most intensely studied cellular signaling networks and has been frequently targeted by the pharmaceutical industry, with more than 30 inhibitors either approved or under clinical evaluation. The ERK–MAPK cascade was originally depicted as a serial and linear, unidirectional pathway that relays extracellular signals, such as mitogenic stimuli, through the cytoplasm to the nucleus. However, we now appreciate that this three-tiered protein kinase cascade is a central core of a complex network with dynamic signaling inputs and outputs and autoregulatory loops. Despite our considerable advances in understanding the ERK–MAPK network, the ability of cancer cells to adapt to the inhibition of key nodes reveals a level of complexity that remains to be fully understood. In this review, we summarize important developments in our understanding of the ERK–MAPK network and identify unresolved issues for ongoing and future study.

Proteomic Profiling of Colon Cancer Tissues: Discovery of New Candidate Biomarkers

Colon cancer is an aggressive tumor form with a poor prognosis. This study reports a comparative proteomic analysis performed by using two-dimensional differential in-gel electrophoresis (2D-DIGE) between 26 pooled colon cancer surgical tissues and adjacent non-tumoral tissues, to identify potential target proteins correlated with carcinogenesis. The DAVID functional classification tool revealed that most of the differentially regulated proteins, acting both intracellularly and extracellularly, concur across multiple cancer steps. The identified protein classes include proteins involved in cell proliferation, apoptosis, metabolic pathways, oxidative stress, cell motility, Ras signal transduction, and cytoskeleton. Interestingly, networks and pathways analysis showed that the identified proteins could be biologically inter-connected to the tumor-host microenvironment, including innate immune response, platelet and neutrophil degranulation, and hemostasis. Finally, transgelin (TAGL), here identified for the first time with four different protein species, collectively down-regulated in colon cancer tissues, emerged as a top-ranked biomarker for colorectal cancer (CRC). In conclusion, our findings revealed a different proteomic profiling in colon cancer tissues characterized by the deregulation of specific pathways involved in hallmarks of cancer. All of these proteins may represent promising novel colon cancer biomarkers and potential therapeutic targets, if validated in larger cohorts of patients.

Dynamic Palmitoylation Events Following T-Cell Receptor Signaling

Palmitoylation is the reversible addition of palmitate to cysteine via a thioester linkage. Following stimulation of the T-cell receptor we find a number of proteins are newly palmitoylated, including those involved in vesicle-mediated transport and Ras signal transduction. Among these stimulation-dependent palmitoylation targets are the v-SNARE VAMP7, important for docking of vesicular LAT during TCR signaling, and the largely undescribed palmitoyl acyltransferase DHHC18 that is expressed in two isoforms in T cells. Using our newly developed On-Plate Palmitoylation Assay (OPPA), we show DHHC18 is capable of palmitoylating VAMP7 at Cys183. Cellular imaging shows that the palmitoylation-deficient protein fails to be retained at the Golgi.

Galectin-1 Is a Novel Structural Component and a Major Regulator of H-Ras Nanoclusters

The organization of Ras proteins into nanoclusters on the inner plasma membrane is essential for Ras signal transduction, but the mechanisms that drive nanoclustering are unknown. Here we show that epidermal growth factor receptor activation stimulates the formation of H-Ras.GTP-Galectin-1 (Gal-1) complexes on the plasma membrane that are then assembled into transient nanoclusters. Gal-1 is therefore an integral structural component of the H-Ras–signaling nanocluster. Increasing Gal-1 levels increases the stability of H-Ras nanoclusters, leading to enhanced effector recruitment and signal output. Elements in the H-Ras C-terminal hypervariable region and an activated G-domain are required for H-Ras–Gal-1 interaction. Palmitoylation is not required for H-Ras–Gal-1 complex formation, but is required to anchor H-Ras–Gal-1 complexes to the plasma membrane. Our data suggest a mechanism for H-Ras nanoclustering that involves a dual role for Gal-1 as a critical scaffolding protein and a molecular chaperone that contributes to H-Ras trafficking by returning depalmitoylated H-Ras to the Golgi complex for repalmitoylation.