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

Abstract

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.

ACKNOWLEDGMENTS

This work was supported by a grant to J.F.H. from the Cancer Research and Prevention Institute of Texas (CPRIT; RP130059), a National Institutes of Health (NIH) Pathway to Independence Award (K99CA188593) to K.-J.C., a grant to A.A.G. from NIH (R01GM100078), Extreme Science and Engineering Discovery Environment (XSEDE; project MCB150054) grants to R.J.C. from the Australian Research Council (DP120100183 and LP120100088) and The University of Queensland, Institute for Molecular Bioscience, and grants to D.E.C. from the University of California, San Diego, Department of Medicine.