K-Ras Promotes Tumorigenicity through Suppression of Non-canonical Wnt Signaling

Wang, Man‐Tzu; Holderfield, Matthew; Galeas, Jacqueline; Delrosario, Reyno; To, Minh D.; Balmain, Allan; McCormick, Frank

doi:10.1016/j.cell.2015.10.041

Cited by 178 publications

(200 citation statements)

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“…Two studies have shown that PKC activators can suppress the growth of K-Ras tumors in nude mice by stimulating K-Ras phosphorylation (39,57). Similarly and consistent with the loss of phosphorylated K-Ras from the PM as a result of prolonged activation of PKG, we observed inhibition of cell proliferation in KRas-positive NSCLC cells upon chronic activation of the AMPK ¡ eNOS ¡ sGC ¡ PKG signaling pathway.…”

Section: Discussionsupporting

confidence: 88%

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

Cho

Casteel

Prakash

et al. 2016

Molecular and Cellular Biology

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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)....

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Section: Discussionsupporting

confidence: 88%

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

Cho

Casteel

Prakash

et al. 2016

Molecular and Cellular Biology

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“…They also showed that diminishing the charge on the polybasic region by stimulating phosphorylation of S181 with bryostatin-1 enhances the ability of PDEδ to solubilize KRAS4b. Recent studies have also shown that KRAS suppresses Wnt/Ca 2+ signaling pathway by direct binding with calmodulin and that the KRAS4b-calmodulin interaction is attenuated by phosphorylation of S181 by PKC (39,40). Our structural data show that six lysine residues present between 175 and 180 do not interact with PDEδ and thus, are not likely to play any role in KRAS4b-PDEδ interaction.…”

Section: Discussionmentioning

confidence: 49%

Structural basis of recognition of farnesylated and methylated KRAS4b by PDEδ

Dharmaiah

Bindu

Tran

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Farnesylation and carboxymethylation of KRAS4b (Kirsten rat sarcoma isoform 4b) are essential for its interaction with the plasma membrane where KRAS-mediated signaling events occur. Phosphodiesterase-δ (PDEδ) binds to KRAS4b and plays an important role in targeting it to cellular membranes. We solved structures of human farnesylated–methylated KRAS4b in complex with PDEδ in two different crystal forms. In these structures, the interaction is driven by the C-terminal amino acids together with the farnesylated and methylated C185 of KRAS4b that binds tightly in the central hydrophobic pocket present in PDEδ. In crystal form II, we see the full-length structure of farnesylated–methylated KRAS4b, including the hypervariable region. Crystal form I reveals structural details of farnesylated–methylated KRAS4b binding to PDEδ, and crystal form II suggests the potential binding mode of geranylgeranylated–methylated KRAS4b to PDEδ. We identified a 5-aa-long sequence motif (Lys-Ser-Lys-Thr-Lys) in KRAS4b that may enable PDEδ to bind both forms of prenylated KRAS4b. Structure and sequence analysis of various prenylated proteins that have been previously tested for binding to PDEδ provides a rationale for why some prenylated proteins, such as KRAS4a, RalA, RalB, and Rac1, do not bind to PDEδ. Comparison of all four available structures of PDEδ complexed with various prenylated proteins/peptides shows the presence of additional interactions due to a larger protein–protein interaction interface in KRAS4b–PDEδ complex. This interface might be exploited for designing an inhibitor with minimal off-target effects.

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“…It was recently suggested that K-ras is the major driver of stem cell or progenitor cell proliferation, while H-ras drives differentiation (75)(76)(77). Therefore, SPRED1's well-documented role in regulating differentiation (2) may integrate in the following way, according to our differential Ras regulation scenario outlined above.…”

Section: Discussionmentioning

confidence: 96%

SPRED1 Interferes with K-ras but Not H-ras Membrane Anchorage and Signaling

Siljamäki

Abankwa

2016

Molecular and Cellular Biology

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The Ras/mitogen-activated protein kinase (MAPK) signaling pathway is tightly controlled by negative feedback regulators, such as the tumor suppressor SPRED1. The SPRED1 gene also carries loss-of-function mutations in the RASopathy Legius syndrome. Growth factor stimulation translocates SPRED1 to the plasma membrane, triggering its inhibitory activity. However, it remains unclear whether SPRED1 there acts at the level of Ras or Raf. We show that pharmacological or galectin-1 (Gal-1)-mediated induction of B-and C-Raf-containing dimers translocates SPRED1 to the plasma membrane. This is facilitated in particular by SPRED1 interaction with B-Raf and, via its N terminus, with Gal-1. The physiological significance of these novel interactions is supported by two Legius syndrome-associated mutations that show diminished binding to both Gal-1 and B-Raf. On the plasma membrane, SPRED1 becomes enriched in acidic membrane domains to specifically perturb membrane organization and extracellular signal-regulated kinase (ERK) signaling of active K-ras4B (here, K-ras) but not H-ras. However, SPRED1 also blocks on the nanoscale the positive effects of Gal-1 on H-ras. Therefore, a combinatorial expression of SPRED1 and Gal-1 potentially regulates specific patterns of K-ras-and H-ras-dependent signaling output. More broadly, our results open up the possibility that related SPRED and Sprouty proteins act in a similar Ras and Raf isoform-specific manner.S PRED (Sprouty-related proteins with an EVH1 domain) proteins are Sprouty-related negative modulators of Ras/mitogen-activated protein kinase (MAPK) signaling, and they have been shown to attenuate extracellular signal-regulated kinase (ERK) activity following various extracellular mitogenic stimuli (e.g., growth factors, cytokines, and chemokines) (1-5). The mammalian SPRED family contains four members, SPRED1, SPRED2, SPRED3, and Eve-3, the last of which is a splice variant of SPRED3 (1, 2, 6). SPRED1 and SPRED2 proteins contain an N-terminal enabled/vasodilator-stimulated phosphoprotein homology-1 (EVH1) domain and a cysteine-rich C-terminal Sprouty-related (SPR) domain, separated by a small c-Kit-binding domain (KBD) (2, 7). The SPR domain is necessary for membrane anchoring following growth factor stimulation (5), and both EVH1 and SPR domains have been implicated in ERK suppression (5,8,9). SPRED3 lacks a functional KBD and shows lower inhibitory activity than SPRED1 and -2, suggesting that the KBD also participates in inhibiting ERK activity (1-5).The precise mechanism of action of how SPRED proteins block MAPK signaling remains unclear. Overexpression of SPRED1 was suggested to increase the recruitment of the Ras effector Raf to the plasma membrane, where its association with Ras does not, however, lead to Raf activation (1, 2, 6). Instead, SPRED1 was reported to prolong Ras-Raf complexation, thus withdrawing Raf from activation by phosphorylation (2, 7). Another study showed that overexpression of SPRED1 inhibits Ras activation, as evidenced by decreased levels of GTP-bound ...

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K-Ras Promotes Tumorigenicity through Suppression of Non-canonical Wnt Signaling

Cited by 178 publications

References 58 publications

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

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

Structural basis of recognition of farnesylated and methylated KRAS4b by PDEδ

SPRED1 Interferes with K-ras but Not H-ras Membrane Anchorage and Signaling

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