Nucleophosmin and Nucleolin Regulate K-Ras Plasma Membrane Interactions and MAPK Signal Transduction

The spatial organization of Ras proteins into nanoclusters on the inner leaflet of the plasma membrane is essential for high fidelity signaling through the MAPK pathway. Here we identify two selective regulators of K-Ras nanoclustering from a proteomic screen for K-Ras interacting proteins. Nucleophosmin (NPM) and nucleolin are predominantly localized to the nucleolus but also have extranuclear functions. We show that a subset of NPM and nucleolin localizes to the inner leaflet of plasma membrane and forms specific complexes with K-Ras but not other Ras isoforms. Active GTP-loaded and inactive GDP-loaded K-Ras both interact with NPM, although NPM-K-Ras binding is increased by growth factor receptor activation. NPM and nucleolin both stabilize K-Ras levels on the plasma membrane, but NPM concurrently increases the clustered fraction of GTP-K-Ras. The increase in nanoclustered GTP-K-Ras in turn enhances signal gain in the MAPK pathway. In summary these results reveal novel extranucleolar functions for NPM and nucleolin as regulators of K-Ras nanocluster formation and activation of the MAPK pathway. The study also identifies a new class of K-Ras nanocluster regulator that operates independently of the structural scaffold galectin-3.Ras proteins are small GTPases that function as molecular switches on the inner leaflet of the plasma membrane, conveying extracellular signals to the cell interior. Ras proteins are critical regulators of signal transduction pathways controlling key cell fates such as cell growth, differentiation, and apoptosis. Deregulation of these pathways results in aberrant cell growth and tumor formation. Mutations that render Ras constitutively active are found in ∼15% of human cancers, making Ras one of the most clinically significant proteins in human carcinogenesis. Oncogenic mutations are most prevalent in the K-Ras gene, accounting for a large proportion of solid tumors including 90% of pancreatic cancer, 50% of colon cancer, and 30% of non-small cell lung cancer (1, 2).The three major Ras isoforms, H-, N-, and K-Ras generate distinct signal outputs in intact cells, signifying specific roles for each isoform. These functional differences stem from significant sequence divergence in the Ras C-terminal 25 amino acids of the hypervariable region (HVR)³ that directs post-translation attachment of different lipid anchors. The minimal membrane anchor of H-Ras comprises two palmitate groups and a farnesyl group, whereas K-Ras is tethered by a farnesyl group and a polybasic domain (3, 4). These minimal anchors, together with flanking protein sequences and the G-domain, interact with lipids and proteins of the plasma membrane, driving the Ras isoforms into spatially and structurally distinct nanodomains on the plasma membrane (5, 6). Ras lateral segregation is further modulated by the activation state of Ras; active GTP-loaded H-Ras is organized in cholesterol-independent nanoclusters, whereas inactive GDP-loaded H-Ras is arrayed in cholesterol-dependent nanoclusters (5, 7–9). Recent work has also shown that GTP-K-Ras clusters into nanodomains that are spatially distinct from GDP-K-Ras, although both types of nanocluster are cholesterol-independent and actin-dependent (7, 9). K-Ras-GTP nanoclustering, however, is regulated by galectin-3, which operates as a nanodomain scaffold (10, 11).Ras-GTP nanoclusters are the sites of Raf/MEK and ERK recruitment to the plasma membrane. Scaffolding all components of the MAPK module within nanoclusters rewires the biochemistry to generate a digital ERKpp output. The operation of Ras-GTP nanoclusters as highly sensitive digital switches is critical to deliver high fidelity signal transmission across the plasma membrane (12–14). A key parameter in epidermal growth factor (EGF) receptor to MAPK signal transmission is the fraction of Ras-GTP that forms nanoclusters; this clustered fraction sets the gain for cellular MAPK signaling (15, 16).NPM (also known as B23) and nucleolin are multifunctional phosphoproteins predominately localized to the nucleolus that play key roles in ribosome biogenesis (17–19). For example, NPM exhibits ribonuclease activity and preferentially cleaves pre-rRNA. NPM and nucleolin also have functions outside of the nucleolus. Both proteins shuttle between the nucleolus and the cytoplasm (20), and this shuttling may allow NPM to operate as molecular chaperone (21). In addition cytosolic NPM is involved in centrosome duplication (22). Like Ras proteins, NPM and nucleolin regulate cell proliferation and transformation and are overexpressed in multiple cancers (23). However, the physiological role of NPM in carcinogenesis remains controversial because it has been described as both an oncogene and a tumor suppressor (23).In this study we identify NPM and nucleolin as proteins that interact specifically with K-Ras but not H-Ras. Furthermore we definitively identify a subset of NPM and nucleolin on the inner leaflet of the plasma membrane where both proteins interact with K-Ras. Importantly, NPM and nucleolin stabilize K-Ras levels on the plasma membrane, leading to an increase in the K-Ras clustered fraction, which amplifies signal output from the MAPK pathway. Combined, our data indicate that NPM and nucleolin play a critical role in signal transduction via the MAPK pathway.