A clathrin-dependent pathway leads to KRas signaling on late endosomes en route to lysosomes

Abstract

Ras proteins are small guanosine triphosphatases involved in the regulation of important cellular functions such as proliferation, differentiation, and apoptosis. Understanding the intracellular trafficking of Ras proteins is crucial to identify novel Ras signaling platforms. In this study, we report that epidermal growth factor triggers Kirsten Ras (KRas) translocation onto endosomal membranes (independently of calmodulin and protein kinase C phosphorylation) through a clathrin-dependent pathway. From early endosomes, KRas but not Harvey Ras or neuroblastoma Ras is sorted and transported to late endosomes (LEs) and lysosomes. Using yellow fluorescent protein–Raf1 and the Raichu-KRas probe, we identified for the first time in vivo–active KRas on Rab7 LEs, eliciting a signal output through Raf1. On these LEs, we also identified the p14–MP1 scaffolding complex and activated extracellular signal-regulated kinase 1/2. Abrogation of lysosomal function leads to a sustained late endosomal mitogen-activated protein kinase signal output. Altogether, this study reveals novel aspects about KRas intracellular trafficking and signaling, shedding new light on the mechanisms controlling Ras regulation in the cell.

Features of Ras activation by a mislocalized oncogenic tyrosine kinase: FLT3 ITD signals via K-Ras at the plasma membrane of Acute Myeloid Leukemia cells

“…Consistent with this notion, partially overlapping localization of sites of intracellular N-Ras activation with an endosomal marker was observed in our study. A similar endocytotic process has been proposed to mediate the accumulation of K-Ras at early endosomes (Lu et al, 2009) and the presence of (active) H-Ras in the endocytic compartment in the context of growth factor signaling has also been documented in a number of cases (Fehrenbacher et al, 2009).…”

Desmoglein-1/Erbin interaction suppresses ERK activation to support epidermal differentiation

“…At the subcellular level, Ras appears in the cytoplasm but also concentrates at the cell periphery, the latter localization becoming more visible in the upper epidermis as the cytoplasmic staining subsides. Punctate, Ras-positive structures in the cytoplasm may represent late endosomes to which both Ras and SHOC2 relocate upon EGF treatment (39,67). As in 3-dimensional cell culture, gt anti-Erbin stained the intermediate layers and appeared primarily localized to cell borders, with cytoplasmic staining also present (Supplemental Figure 3, B and C).…”

Establishment of Extracellular Signal-Regulated Kinase 1/2 Bistability and Sustained Activation through Sprouty 2 and Its Relevance for Epithelial Function

“…One of the determinants of asthma severity is Mice were immunized and exposed to a combination of three allergens, dust mites, ragweed, and Aspergillus (DRA), or saline as described previously (31). Three weeks after the last allergen exposure the mice were examined for pERK1/2 immunostaining (green).…”

“…(D to F) Endosomal pERK1/2 in human asthma. (D) Lung biopsy samples obtained from 6 asthmatic subjects were immunostained for pERK1/2 and counterstained with DAPI (pseudocolored red) as described previously (31). A representative airway epithelial image (magnification, ϫ100) is shown.…”