AMPK and Endothelial Nitric Oxide Synthase Signaling Regulates K-Ras Plasma Membrane Interactions via Cyclic GMP-Dependent Protein Kinase 2

Abstract

f K-Ras must localize to the plasma membrane and be arrayed in nanoclusters for biological activity. We show here that K-Ras is a substrate for cyclic GMP-dependent protein kinases (PKGs). In intact cells, activated PKG2 selectively colocalizes with K-Ras on the plasma membrane and phosphorylates K-Ras at Ser181 in the C-terminal polybasic domain. K-Ras phosphorylation by PKG2 is triggered by activation of AMP-activated protein kinase (AMPK) and requires endothelial nitric oxide synthase and soluble guanylyl cyclase. Phosphorylated K-Ras reorganizes into distinct nanoclusters that retune the signal output. Phosphorylation acutely enhances K-Ras plasma membrane affinity, but phosphorylated K-Ras is progressively lost from the plasma membrane via endocytic recycling. Concordantly, chronic pharmacological activation of AMPK ¡ PKG2 signaling with mitochondrial inhibitors, nitric oxide, or sildenafil inhibits proliferation of K-Ras-positive non-small cell lung cancer cells. The study shows that K-Ras is a target of a metabolic stress-signaling pathway that can be leveraged to inhibit oncogenic K-Ras function. Ras proteins are small GTPases that regulate important cellular signaling cascades to control cell growth, proliferation, and differentiation (1). The three Ras isoforms, H-, N-, and K-Ras4B (here, K-Ras), are ubiquitously expressed in mammalian cells. Ras proteins must be localized to the inner leaflet of the plasma membrane (PM) by a C-terminal membrane anchor for biological activity. In the case of K-Ras, the anchor comprises a posttranslationally attached C-terminal cysteine farnesyl-methyl ester operating in concert with a polybasic motif of 6 lysine residues (2, 3). Electrostatic interactions between the K-Ras C-terminal polybasic domain and the negatively charged inner leaflet of the PM provide membrane affinity (2, 4-9). Maintenance of K-Ras on the PM also requires the chaperone protein PDE␦ (10). Cytosolic PDE␦ binds K-Ras released from the PM as a result of endocytosis and unloads K-Ras in the perinuclear region in response to Arl2/3 binding, whence K-Ras translocates to the recycling endosome (RE) for redelivery to the PM by vesicular transport (11). Ras proteins on the PM are spatially organized into nanodomains, called nanoclusters, that are required for high-fidelity signal transduction by the Ras/mitogen-activated protein kinase (MAPK) pathway (12-14). Ras GTP nanoclusters contain ϳ6 to 7 Ras proteins, are Ͻ20 nm in diameter, and are exclusive platforms for Raf recruitment and MEK/extracellular signal-regulated kinase (ERK) activation. Perturbation of the spatiotemporal dynamics of Ras nanoclustering disrupts cellular signaling (15, 16).We have used a high-content cell-based screen (HCS) to identify multiple chemical compounds that mislocalize K-Ras from the PM and abrogate K-Ras signal transmission (17,18). One group of compounds disrupts the cellular phosphatidylserine (PtdSer) distribution or PtdSer levels with a consequent reduction in the PtdSer content of the inner leaflet of the PM (18-21)....

Lipid-Sorting Specificity Encoded in K-Ras Membrane Anchor Regulates Signal Output

“…FRET efficiencies followed the same trend as the corresponding LactC2 LBI values (Figure S3J). Phosphorylation of Ser181 by protein kinase C (PKC) (Bivona et al, 2006) or protein kinase G (PKG) (Cho et al, 2016) also reduces the net charge on the K-Ras PBD. We therefore conducted lipid mapping of RFP-K-RasG12V nanoclusters after acute activation of PKG.…”

Deciphering lipid codes: K‐Ras as a paradigm

“…K-Ras can be phosphorylated on serine (Ser) 181, adjacent to the PBD, by protein kinase C (PKC) or protein kinase G (isoform PKG2) 45,57 . K-Ras phosphorylation causes mis-localization of K-Ras from the PM and compromises K-Ras-dependent cancer cell proliferation 58 .…”

“…57,58 In addition to PKC, PKG2 can robustly phosphorylate K-Ras on Ser181 as the target of an AMPK regulated signaling pathway. 45 Thus, pharmacological activation of the AMPK, eNOS, sGC, PKG2 pathway at multiple levels, with agents such as DEA-NO, oligomycin, [64][65][66][67] AICAR, sildenafil or metformin all mis-localize K-Ras from the PM and effectively inhibit the proliferation of K-Ras transformed NSCLC cell lines where this PKG2 signaling pathway is intact. 45 K-Ras does not co-localize with PIP 2 in intact PM and K-Ras nanoclustering and PM binding are independent of PIP 2 in lipid addback experiments.…”

“…The Ras-clustered fraction is a system parameter that sets signal gain. Thus, perturbations of the PM lipid environment that reduce Ras-GTP nanoclustering [28][29][30][31][43][44][45][46] will decrease the clustered fraction and compromise signal output, even if total number of active Ras molecules on the PM stays the same. Simply put, fewer nanoclusters !…”

Posttranslational Modifications of RAS Proteins

“…By analogy, KRAS4B harbors a farnesyl-electrostatic switch. Serine 181 of KRAS4B is a substrate for both protein kinase Cs (PKCs) (107, 109) and cyclic GMP-dependent protein kinases 2 (PKG2) (110). PKG2 is activated to phosphorylate KRAS4B downstream of AMP-activated protein kinase (AMPK) and requires endothelial nitric oxide synthase and soluble guanylyl cyclase.…”