Ubiquitination of K-Ras Enhances Activation and Facilitates Binding to Select Downstream Effectors

Sasaki, Atsuo T.; Carracedo, Arkaitz; Locasale, Jason W.; Anastasiou, Dimitrios; Takeuchi, Koh; Kahoud, Emily Rose; Haviv, Sasson; Asara, John M.; Pandolfi, Pier Paolo; Cantley, Lewis C.

doi:10.1126/scisignal.2001518

Cited by 163 publications

(200 citation statements)

References 30 publications

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“…Additionally, several lysines within the core G domain of RAS undergo post-translational modifications, including acetylation, ubiquitylation, and methylation (5), but the role of these distinct modifications in regulating RAS function is still unclear. For example, KRAS monoubiquitylation at lysine 147 up-regulates RAS activity, signaling, and tumorigenesis (6). Additionally, lysine 104 has been shown to be a minor site of ubiquitylation, and we have previously shown that ubiquitylation of KRAS at this position does not alter the intrinsic biochemical properties or regulation by GEFs and GAPs (7).…”

mentioning

confidence: 99%

A KRAS GTPase K104Q Mutant Retains Downstream Signaling by Offsetting Defects in Regulation

Yin

Kistler

George

et al. 2017

Journal of Biological Chemistry

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Edited by Norma AllewellThe KRAS GTPase plays a critical role in the control of cellular growth. The activity of KRAS is regulated by guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), and also post-translational modification. Lysine 104 in KRAS can be modified by ubiquitylation and acetylation, but the role of this residue in intrinsic KRAS function has not been well characterized. We find that lysine 104 is important for GEF recognition, because mutations at this position impaired GEF-mediated nucleotide exchange. Because the KRAS K104Q mutant has recently been employed as an acetylation mimetic, we conducted a series of studies to evaluate its in vitro and cellbased properties. Herein, we found that KRAS K104Q exhibited defects in both GEF-mediated exchange and GAP-mediated GTP hydrolysis, consistent with NMR-detected structural perturbations in localized regions of KRAS important for recognition of these regulatory proteins. Despite the partial defect in both GEF and GAP regulation, KRAS K104Q did not alter steady-state GTP-bound levels or the ability of the oncogenic KRAS G12V mutant to cause morphologic transformation of NIH 3T3 mouse fibroblasts and of WT KRAS to rescue the growth defect of mouse embryonic fibroblasts deficient in all Ras genes. We conclude that the KRAS K104Q mutant retains both WT and mutant KRAS function, probably due to offsetting defects in recognition of factors that up-regulate (GEF) and down-regulate (GAP) RAS activity.RAS proteins function as molecular switches that cycle between active GTP-and inactive GDP-bound states to regulate signal transduction pathways that modulate cellular growth control. In the unstimulated cell, RAS proteins are populated in their inactive GDP-bound state. However, in response to growth-stimulatory signals, guanine nucleotide exchange factors (GEFs) 2 co-localize and up-regulate RAS by facilitating exchange of GDP for GTP. Inactivation of RAS is achieved through GTPase-activating proteins (GAPs) that bind to GTPbound RAS and promote GTP hydrolysis (1, 2). Several point mutations in RAS have been identified that dysregulate RAS nucleotide exchange or hydrolysis, often leading to hyperactivation and promoting tumorigenesis. The most common RAS mutations identified in cancer occur at residues 12, 13, and 61 and render RAS GAP defective, thereby populating RAS in its active GTP-bound state (3). Constitutive hyperactivation of RAS promotes chronic stimulation of effector-mediated downstream pathways, causing deregulated growth and tumorigenic growth transformation.RAS contains two dynamic regions termed switch I (SWI; residues 30 -37) and switch II (SWII; residues 60 -76 with 66 -74 corresponding to helix 2 (H2)) that populate distinct conformations when the protein is bound to GDP versus GTP. Effectors and GAP proteins recognize specific conformations of the switch regions and bind with preferential affinity to the active GTP-bound state. Activated GTP-bound RAS can interact with multiple effectors (e.g. RAF kinase, RAL exchang...

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confidence: 99%

A KRAS GTPase K104Q Mutant Retains Downstream Signaling by Offsetting Defects in Regulation

Yin

Kistler

George

et al. 2017

Journal of Biological Chemistry

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“…Although no function has yet been assigned to monoubiquitination of Rac1, it is possible that this modification is involved in the mechanism by which Rho interacts with downstream effectors. Monoubiquitination of mammalian Ras proteins can lead to cell transformation (71) and does so by promoting nucleotide exchange or by impeding the binding of the GTPase activating protein (72,73).…”

Section: Discussionmentioning

confidence: 99%

Guanine Nucleotide-binding Protein (Gα) Endocytosis by a Cascade of Ubiquitin Binding Domain Proteins Is Required for Sustained Morphogenesis and Proper Mating in Yeast

Dixit¹,

Baker²,

Sacks³

et al. 2014

Journal of Biological Chemistry

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Background:The yeast G␣ protein contains a unique domain that is monoubiquitinated, leading to vacuolar degradation. Results: A gene deletion screen reveals ubiquitin binding domain proteins necessary for G␣ trafficking. Loss of the ubiquitination domain impedes cellular morphogenesis and mating. Conclusion: Proper endocytosis of G␣ is required for sustained morphogenesis and efficient mating. Significance: G␣ endocytosis promotes signaling. Heterotrimeric G proteins are well known to transmit signals from cell surface receptors to intracellular effector proteins.There is growing appreciation that G proteins are also present at endomembrane compartments, where they can potentially interact with a distinct set of signaling proteins. Here, we examine the cellular trafficking function of the G protein ␣ subunit in yeast, Gpa1. Gpa1 contains a unique 109-amino acid insert within the ␣-helical domain that undergoes a variety of posttranslational modifications. Among these is monoubiquitination, catalyzed by the NEDD4 family ubiquitin ligase Rsp5. Using a newly optimized method for G protein purification together with biophysical measures of structure and function, we show that the ubiquitination domain does not influence enzyme activity. By screening a panel of 39 gene deletion mutants, each lacking a different ubiquitin binding domain protein, we identify seven that are necessary to deliver Gpa1 to the vacuole compartment including four proteins (Ede1, Bul1, Ddi1, and Rup1) previously not known to be involved in this process. Finally, we show that proper endocytosis of the G protein is needed for sustained cellular morphogenesis and mating in response to pheromone stimulation. We conclude that a cascade of ubiquitin-binding proteins serves to deliver the G protein to its final destination within the cell. In this instance and in contrast to the previously characterized visual system, endocytosis from the plasma membrane is needed for proper signal transduction rather than for signal desensitization.G␣ proteins are enzymatic switches that are part of a multicomponent signaling complex. The complex typically consists of a seven-transmembrane G protein-coupled receptor, a guanine nucleotide-binding protein (G␣), and an associated dimer consisting of ␤ and ␥ subunits (G␤␥) (1). Signaling is turned on and off based on receptor activation, which in turn dictates the nucleotide-bound state of the G␣ protein. When G␣ is GDPbound, G␤␥ is sequestered, and signaling pathways are off (1). When G␣ releases GDP and binds GTP, G␤␥ dissociates, and the signaling pathways are turned on. Subsequent GTP hydrolysis is accelerated by regulators of G protein signaling (RGS 3 proteins) (2, 3). Large G␣ proteins contain a Ras-like domain as well as an independently folded ␣-helical domain (1). Within this group of proteins there is a well established role for the Ras-like domain in specifying interactions with G␤␥, effectors, and RGS proteins (1). However, recent evidence has shown that the ␣-helical domain is also important for signa...

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“…2010), whereas monoubiquitylation of K‐Ras at K104 or K147 by an unknown E3 ligase does not affect its intracellular localization but rather promotes the binding and activation of the downstream effector proteins RAF and phosphoinositide 3‐kinase by an unknown mechanism (Sasaki et al . 2011). …”

Section: Regulation Of Membrane‐associated Processes By Monoubiquitylmentioning

confidence: 99%

Protein monoubiquitylation: targets and diverse functions

Nakagawa

Nakayama

2015

Genes to Cells

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Ubiquitin is a 76‐amino acid protein whose conjugation to protein targets is a form of post‐translational modification. Protein ubiquitylation is characterized by the covalent attachment of the COOH‐terminal carboxyl group of ubiquitin to an amino group of the substrate protein. Given that the NH2‐terminal amino group is usually masked, internal lysine residues are most often targeted for ubiquitylation. Polyubiquitylation refers to the formation of a polyubiquitin chain on the substrate as a result of the ubiquitylation of conjugated ubiquitin. The structures of such polyubiquitin chains depend on the specific lysine residues of ubiquitin targeted for ubiquitylation. Most of the polyubiquitin chains other than those linked via lysine‐63 and methionine‐1 of ubiquitin are recognized by the proteasome and serve as a trigger for substrate degradation. In contrast, polyubiquitin chains linked via lysine‐63 and methionine‐1 serve as a binding platform for proteins that function in immune signal transduction or DNA repair. With the exception of a few targets such as histones, the functions of protein monoubiquitylation have remained less clear. However, recent proteomics analysis has shown that monoubiquitylation occurs more frequently than polyubiquitylation, and studies are beginning to provide insight into its biologically important functions. Here, we summarize recent findings on protein monoubiquitylation to provide an overview of the targets and molecular functions of this modification.

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Ubiquitination of K-Ras Enhances Activation and Facilitates Binding to Select Downstream Effectors

Cited by 163 publications

References 30 publications

A KRAS GTPase K104Q Mutant Retains Downstream Signaling by Offsetting Defects in Regulation

A KRAS GTPase K104Q Mutant Retains Downstream Signaling by Offsetting Defects in Regulation

Guanine Nucleotide-binding Protein (Gα) Endocytosis by a Cascade of Ubiquitin Binding Domain Proteins Is Required for Sustained Morphogenesis and Proper Mating in Yeast

Protein monoubiquitylation: targets and diverse functions

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