Galectin-8 binds to the Farnesylated C-terminus of K-Ras4B and Modifies Ras/ERK Signaling and Migration in Pancreatic and Lung Carcinoma Cells.

Christopher Meinohl,Marisa Heipel,Stefanie Wirth,Sarah J Barnard,Katja Becker,Klaudia Giehl,Andre Menke,Johannes Madlung,Andreea Porr,Monika Unger,Karin Fritz-Wolf,Alfred Nordheim

doi:10.3390/cancers12010030

Abstract

K-Ras is the most prominent driver of oncogenesis and no effective K-Ras inhibitors have been established despite decades of intensive research. Identifying new K-Ras-binding proteins and their interaction domains offers the opportunity for defining new approaches in tackling oncogenic K-Ras. We have identified Galectin-8 as a novel, direct binding protein for K-Ras4B by mass spectrometry analyses and protein interaction studies. Galectin-8 is a tandem-repeat Galectin and it is widely expressed in lung and pancreatic carcinoma cells. siRNA-mediated depletion of Galectin-8 resulted in increased K-Ras4B content and ERK1/2 activity in lung and pancreatic carcinoma cells. Moreover, cell migration and cell proliferation were inhibited by the depletion of Galectin-8. The K-Ras4B–Galectin-8 interaction is indispensably associated with the farnesylation of K-Ras4B. The lysine-rich polybasic domain (PBD), a region that is unique for K-Ras4B as compared to H- and N-Ras, stabilizes the interaction and accounts for the specificity. Binding assays with the deletion mutants of Galectin-8, comprising either of the two carbohydrate recognition domains (CRD), revealed that K-Ras4B only interacts with the N-CRD, but not with the C-CRD. Structural modeling uncovers a potential binding pocket for the hydrophobic farnesyl chain of K-Ras4B and a cluster of negatively charged amino acids for interaction with the positively charged lysine residues in the N-CRD. Our results demonstrate that Galectin-8 is a new binding partner for K-Ras4B and it interacts via the N-CRD with the farnesylated PBD of K-Ras, thereby modulating the K-Ras effector pathways as well as cell proliferation and migration.

Highlights

Monomeric GTPases of the Ras family act as molecular switches of signaling pathways, thereby modulating many aspects of physiologic and pathophysiologic cell behavior, including cell proliferation, differentiation, survival, and apoptosis, as well as cell migration and invasion
Our binding studies show that the farnesyl moiety of K-Ras and its polybasic region is indispensable for the complex formation with Galectin-8 via the N-carbohydrate recognition domain (CRD); we suggest that a farnesyl binding pocket in Galectin-8 must exist in conjunction with a negative cluster of amino acids that interact with the positively charged lysine stretch of K-Ras
The biochemical and structural characterization of the K-Ras-Galectin-8 interacting domains revealed that the binding is mediated via the farnesyl moiety of K-Ras and is highly supported by the polybasic stretch of lysine residues in the C-terminus of K-Ras

Summary

Introduction

Monomeric GTPases of the Ras family act as molecular switches of signaling pathways, thereby modulating many aspects of physiologic and pathophysiologic cell behavior, including cell proliferation, differentiation, survival, and apoptosis, as well as cell migration and invasion. They are the most frequently mutated human oncogenes in cancer and are most prominently associated with human malignancies. Missense gain of function mutations, especially in codon 12, 13, or 61, render Ras constitutively active in its GTP-bound state These mutations are found in 27% of all human cancer, and KRAS is most commonly affected in pancreatic (with around 90%), colon (40%), and lung (25%) adenocarcinoma [1]. K-Ras4A is less abundant and far less studied than K-Ras4B [2,3]

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