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

Kerry L Inder,Chiyan Lau,Dorothy Loo,Natasha Chaudhary,Andrew Goodall,Sally Martin,Alun Jones,Dharini Van Der Hoeven,Robert G Parton,Michelle M Hill,John F Hancock

doi:10.1074/jbc.m109.001537

Abstract

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.

Highlights

The three major Ras isoforms, H, N, and K-Ras generate distinct signal outputs in intact cells, signifying specific roles for each isoform
We definitively identify a subset of NPM and nucleolin on the inner leaflet of the plasma membrane where both proteins interact with K-Ras
Ras proteins are laterally segregated into transient nanodomains, a distribution that is essential for high fidelity signal transduction via the MAPK pathway [13, 14]

Summary

EXPERIMENTAL PROCEDURES

Plasmids—GST-CTH (166 –189) and GST-CTK (166 –188) were attained by subcloning CTH and CTK nucleotide sequence into pFASTBAC-DUAL transfer vector (Invitrogen). Recombinant bacmids were transfected into Sf9 cells by the Protein Expression Facility of the University of Queensland. Affinity Chromatography—GST fusion proteins were purified from Sf9 cell lysates by binding to glutathione-agarose beads (Sigma); washed extensively in 1 M KCl, 1% Triton X-100, 1 mM dithiothreitol; and equilibrated three times with 10 mM Tris-HCl (pH 7.5), 25 mM KCl, 2.5 mM CaCl2. The beads were washed ten times for 5 min with buffer containing 100 mM KCl, 10 mM Tris-Cl, 2.5 mM CaCl2, and 1 mM dithiothreitol. The threshold criteria were set as follows: mass range, 850 – 4000 Da; minimum cluster area, 500; minimum signal-tonoise, 20; maximum number of ms/ms spectra per spot, 10. The cells grown on coverslips were washed once in phosphate-buffered saline and incubated in poly-L-lysine for 1 min. The coverslips were washed three times for 5 s in 1/3

Buffer A and then briefly in buffer

Our results so far indicate that the

Findings

DISCUSSION