Determining the ERK-regulated phosphoproteome driving KRAS-mutant cancer.

To delineate the mechanisms by which the ERK1 and ERK2 mitogen-activated protein kinases support mutant KRAS-driven cancer growth, we determined the ERK-dependent phosphoproteome in KRAS-mutant pancreatic cancer. We determined that ERK1 and ERK2 share near-identical signaling and transforming outputs and that the KRAS-regulated phosphoproteome is driven nearly completely by ERK. We identified 4666 ERK-dependent phosphosites on 2123 proteins, of which 79 and 66%, respectively, were not previously associated with ERK, substantially expanding the depth and breadth of ERK-dependent phosphorylation events and revealing a considerably more complex function for ERK in cancer.

Functional and biological heterogeneity of KRAS mutations.

Missense mutations at the three hotspots in the guanosine triphosphatase (GTPase) RAS-Gly, Gly, and Gln (commonly known as G12, G13, and Q61, respectively)-occur differentially among the three RAS isoforms. Q61 mutations in are infrequent and differ markedly in occurrence. Q61H is the predominant mutant (at 57%), followed by Q61R/L/K (collectively 40%), and Q61P and Q61E are the rarest (2 and 1%, respectively).

RHOA takes the RHOad less traveled to cancer.

RAS and RHO GTPases function as signaling nodes that regulate diverse cellular processes. Whereas RAS mutations were identified in human cancers nearly four decades ago, only recently have mutations in two RHO GTPases, RAC1 and RHOA, been identified in cancer. RAS mutations are found in a diverse spectrum of human cancer types.

Concurrent Inhibition of ERK and Farnesyltransferase Suppresses the Growth of HRAS Mutant Head and Neck Squamous Cell Carcinoma.

Human papilloma virus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) is a common cancer worldwide with an unmet need for more effective, less toxic treatments. Currently, both the disease and the treatment of HNSCC cause significant mortality and morbidity. Targeted therapies hold new promise for patients with HPV-negative status whose tumors harbor oncogenic HRAS mutations.

Aberrant Expression and Subcellular Localization of ECT2 Drives Colorectal Cancer Progression and Growth.

ECT2 is an activator of RHO GTPases that is essential for cytokinesis. In addition, ECT2 was identified as an oncoprotein when expressed ectopically in NIH/3T3 fibroblasts. However, oncogenic activation of ECT2 resulted from N-terminal truncation, and such truncated ECT2 proteins have not been found in patients with cancer.

Targeting p130Cas- and microtubule-dependent MYC regulation sensitizes pancreatic cancer to ERK MAPK inhibition.

To identify therapeutic targets for KRAS mutant pancreatic cancer, we conduct a druggable genome small interfering RNA (siRNA) screen and determine that suppression of BCAR1 sensitizes pancreatic cancer cells to ERK inhibition. Integrative analysis of genome-scale CRISPR-Cas9 screens also identify BCAR1 as a top synthetic lethal interactor with mutant KRAS. BCAR1 encodes the SRC substrate p130Cas.

RAS and RHO family GTPase mutations in cancer: twin sons of different mothers?

The RAS and RHO family comprise two major branches of the RAS superfamily of small GTPases. These proteins function as regulated molecular switches and control cytoplasmic signaling networks that regulate a diversity of cellular processes, including cell proliferation and cell migration. In the early 1980s, mutationally activated genes encoding KRAS, HRAS and NRAS were discovered in human cancer and now comprise the most frequently mutated oncogene family in cancer.

The molecular basis for immune dysregulation by the hyperactivated E62K mutant of the GTPase RAC2.

The RAS-related C3 botulinum toxin substrate 2 (RAC2) is a member of the RHO subclass of RAS superfamily GTPases required for proper immune function. An activating mutation in a key switch II region of RAC2 (RAC2) involved in recognizing modulatory factors and effectors has been identified in patients with common variable immune deficiency. To better understand how the mutation dysregulates RAC2 function, we evaluated the structure and stability, guanine nucleotide exchange factor (GEF) and GTPase-activating protein (GAP) activity, and effector binding of RAC2 Our findings indicate the E62K mutation does not alter RAC2 structure or stability.

Gain-of-Function Mutations Promote Focal Adhesion Kinase Activation and Dependency in Diffuse Gastric Cancer.

Diffuse gastric cancer (DGC) is a lethal malignancy lacking effective systemic therapy. Among the most provocative recent results in DGC has been that of highly recurrent missense mutations in the GTPase RHOA. The function of these mutations has remained unresolved.

Inflammation-Sensitive Myosin-X Functionally Supports Leukocyte Extravasation by Cdc42-Mediated ICAM-1-Rich Endothelial Filopodia Formation.

Leukocyte transendothelial migration is key to inflammation. Leukocytes first start rolling over the inflamed endothelium, followed by firmly adhering to it. Under inflammatory conditions, endothelial cells express small finger-like protrusions that stick out into the lumen.

Endothelial CD2AP Binds the Receptor ICAM-1 To Control Mechanosignaling, Leukocyte Adhesion, and the Route of Leukocyte Diapedesis In Vitro.

Inflammation is driven by excessive transmigration (diapedesis) of leukocytes from the blood to the tissue across the endothelial cell monolayer that lines blood vessels. Leukocyte adhesion, crawling, and transmigration are regulated by clustering of the endothelial mechanosensitive receptor intercellular adhesion molecule-1 (ICAM-1). Whereas several proteins are known to promote ICAM-1 function, the molecular mechanisms that limit ICAM-1-mediated adhesion to prevent excessive leukocyte transmigration remain unknown.

A local VE-cadherin and Trio-based signaling complex stabilizes endothelial junctions through Rac1.

Endothelial cell-cell junctions maintain a restrictive barrier that is tightly regulated to allow dynamic responses to permeability-inducing angiogenic factors, as well as to inflammatory agents and adherent leukocytes. The ability of these stimuli to transiently remodel adherens junctions depends on Rho-GTPase-controlled cytoskeletal rearrangements. How the activity of Rho-GTPases is spatio-temporally controlled at endothelial adherens junctions by guanine-nucleotide exchange factors (GEFs) is incompletely understood.

Cell-stiffness-induced mechanosignaling - a key driver of leukocyte transendothelial migration.

The breaching of cellular and structural barriers by migrating cells is a driving factor in development, inflammation and tumor cell metastasis. One of the most extensively studied examples is the extravasation of activated leukocytes across the vascular endothelium, the inner lining of blood vessels. Each step of this leukocyte transendothelial migration (TEM) process is regulated by distinct endothelial adhesion receptors such as the intercellular adhesion molecule 1 (ICAM1).

Toward understanding RhoGTPase specificity: structure, function and local activation.

Cell adhesion and migration are regulated through the concerted action of cytoskeletal dynamics and adhesion proteins, the activity of which is governed by RhoGTPases. Specific RhoGTPase signaling requires spatio-temporal activation and coordination of subsequent protein-protein and protein-lipid interactions. The nature, location and duration of these interactions are dependent on polarized extracellular triggers, such as cell-cell contact, and intracellular modifying events, such as phosphorylation.

Actin-binding proteins differentially regulate endothelial cell stiffness, ICAM-1 function and neutrophil transmigration.

Chronic vascular inflammation is driven by interactions between activated leukocytes and the endothelium. Leukocyte β2-integrins bind to endothelial intercellular adhesion molecule 1 (ICAM-1), which allows leukocyte spreading, crawling and transendothelial migration. Leukocytes scan the vascular endothelium for permissive sites to transmigrate, which suggests that there is apical membrane heterogeneity within the endothelium.

The human minor histocompatibility antigen 1 is a RhoGAP.

The human minor Histocompatibility Antigen HMHA-1 is a major target of immune responses after allogeneic stem cell transplantation applied for the treatment of leukemia and solid tumors. The restriction of its expression to hematopoietic cells and many solid tumors raised questions regarding its cellular functions. Sequence analysis of the HMHA-1 encoding HMHA1 protein revealed the presence of a possible C-terminal RhoGTPase Activating Protein (GAP) domain and an N-terminal BAR domain.

Ubiquitin links to cytoskeletal dynamics, cell adhesion and migration.

Post-translational modifications are used by cells to link additional information to proteins. Most modifications are subtle and concern small moieties such as a phosphate group or a lipid. In contrast, protein ubiquitylation entails the covalent attachment of a full-length protein such as ubiquitin.

Dimeric plant RhoGAPs are regulated by its CRIB effector motif to stimulate a sequential GTP hydrolysis.

RopGAPs are GTPase-activating proteins (GAPs) for plant Rho proteins (ROPs). The largest RopGAP family is characterized by the plant-specific combination of a classical RhoGAP domain and a Cdc42/Rac interactive binding (CRIB) motif, which, in animal and fungi, has never been found in GAPs but in effectors for Cdc42 and Rac1. Very little is known about the molecular mechanism of the RopGAP activity including the regulatory role of the CRIB motif.

The unique plant RhoGAPs are dimeric and contain a CRIB motif required for affinity and specificity towards cognate small G proteins.

Plant Rho proteins (ROPs) are inactivated by specific GTPase activating proteins, called RopGAPs. Many of these comprise the exclusive combination of a classic, catalytic Arg-containing RhoGAP domain, and a Cdc42/ Rac interactive binding (CRIB) motif which in animal and fungi has been identified in effectors for Cdc42 and Rac1, but never in any GAP protein. Both elements are required for an efficient RopGAP activity.

Rho proteins of plants--functional cycle and regulation of cytoskeletal dynamics.

Rho-related ROP proteins are molecular switches that essentially regulate a wide variety of processes. Of central interest is their influence on the plant cytoskeleton by which they affect vital processes like cell division, growth, morphogenesis, and pathogen defense. ROPs switch between GTP- and GDP-bound conformations by strictly regulated nucleotide exchange and GTP-hydrolysis, and only the active GTP-form interacts with downstream effectors to ultimately provoke a biological response.

A genomic screen identifies Dsk2p and Rad23p as essential components of ER-associated degradation.

We developed a growth test to screen for yeast mutants defective in endoplasmic reticulum (ER) quality control and associated protein degradation (ERAD) using the membrane protein CTL*, a chimeric derivative of the classical ER degradation substrate CPY*. In a genomic screen of approximately 5,000 viable yeast deletion mutants, we identified genes necessary for ER quality control and degradation. Among the new gene products, we identified Dsk2p and Rad23p.