Galectin-3 Augments K-Ras Activation and Triggers a Ras Signal That Attenuates ERK but Not Phosphoinositide 3-Kinase Activity

Abstract

Depending on the cellular context, Ras can activate characteristic effectors by mechanisms still poorly understood. Promotion by galectin-1 of Ras activation of Raf-1 but not of phosphoinositide 3-kinase (PI3-K) is one such mechanism. In this report, we describe a mechanism controlling selectivity of K-Ras4B (K-Ras), the most important Ras oncoprotein. We show that galectin-3 acts as a selective binding partner of activated K-Ras. Galectin-3 co-immunoprecipitated significantly better with KRas-GTP than with K-Ras-GDP, H-Ras, or N-Ras and colocalized with green fluorescent protein-K-Ras(G12V), not with green fluorescent protein-H-Ras(G12V), in the cell membrane. Co-transfectants of K-Ras/galectin-3, but not of H-Ras/galectin-3, exhibited enhanced and prolonged epidermal growth factor-stimulated increases in Ras-GTP, Raf-1 activity, and PI3-K activity. Extracellular signal-regulated kinase (ERK) activity, however, was attenuated in K-Ras/galectin-3 and in K-Ras(G12V)/galectin-3 co-transfectants. Galectin-3 antisense RNA inhibited the epidermal growth factor-stimulated increase in K-Ras-GTP but enhanced ERK activation and augmented K-Ras(G12V) transformation activity. Thus, unlike galectin-1, which prolongs Ras activation of ERK and inhibits PI3-K, K-Ras-GTP/galectin-3 interactions promote, in addition to PI3-K and Raf-1 activation, a third inhibitory signal that attenuates active ERK. These experiments established a novel and specific mechanism controlling the duration and selectivity of signals of active K-Ras, which is extremely important in many human tumors.

The Ras‐ERK pathway: Understanding site‐specific signaling provides hope of new anti‐tumor therapies

“…[35] Galectin-3 functions in a similar way, but specifically for K-Ras. [36] These ancillary proteins are critical for the formation of nanoclusters: antagonizing Galectin-1 results in the displacement of H-Ras from the PM and in the inhibition of Ras biological activity. [37] Galectin-1 not only behaves as a key scaffold for the formation of H-Ras nanoclusters, it also functions as a molecular chaperone that contributes to H-Ras trafficking, by returning depalmitoylated H-Ras to the GC.…”

“…[52] Effector usage can be influenced by site-specific regulatory proteins, as exemplified by Galectins: Galectin-1 diverts H-Ras signals to Raf-1 at the expense of PI3K, [38] whereas Galectin-3 attenuates ERK activation without affecting PI3K activity. [36] Ras trafficking can also impact on effector utilization. Ubiquitination of H-and N-Ras promotes their association to endosomes, where reduced availability of Raf results in diminished ERK activation.…”

“…The case is not so clear with Galectin-3, as its down-regulation potentiates ERK activation and K-RasV12-induced transformation. [36] ERK scaffold proteins are indeed candidates with an enormous potential to become site-specific therapeutic targets. As mentioned before, they recently emerged as important spacerestricted modulators of ERKs signals.…”

The Ras–Membrane Interface: Isoform-Specific Differences in the Catalytic Domain

“…Galectin-1 and galectin-3 help stabilize H-RasGTP and K-RasGTP, respectively, when each isoform is localized in the correct lipid microdomain (discussed below) to promote high fidelity nanoclustering (13). The hydrophobic pocket of galectin is proposed to interact with the farnesyl group on Ras, potentially stabilizing the protein in the appropriate membrane orientation needed for a specific Ras-effector interaction (14). Although overexpression of galectins can increase Ras nanocluster output and has important implications in oncogenesis (13), proteins that bind the farnesyl group, such as phosphodiesterase-d and GDP dissociation inhibitors (GDI), can act as an "off-switch" for small GTPases by sequestering them from the membrane (15).…”

The Two Faces of Galectin-3: Roles in Various Pathological Conditions

“…It has been demonstrated that Galectin-3 favours a broad range of cancer cell activities, such as malignant cell transformation and tumour growth (62)(63)(64), cell adhesion, migration and invasion (65-69), anoikis resistance (70), apoptosis inhibition (71), (72) and angiogenesis (73).…”