Site-specific monoubiquitination activates Ras by impeding GTPase-activating protein function

Baker, Rachael; Lewis, Steven M.; Sasaki, Atsuo T.; Wilkerson, Emily M.; Locasale, Jason W.; Cantley, Lewis C.; Kuhlman, Brian; Dohlman, Henrik G.; Campbell, Sharon L.

doi:10.1038/nsmb.2430

Cited by 82 publications

(138 citation statements)

References 44 publications

(63 reference statements)

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“…The catalytic domains of human SOS1 (SOS cat , residues 566 -1049) (26) and p120-RASGAP (GAP cat , residues 764 -981) were expressed in a pQlinkH vector (Addgene) and purified as described previously (33). cDNA sequences encoding the isolated RBDs of human BRAF (amino acids 149 -232) and CRAF (amino acids 51-132) were subcloned in a pET28a bacterial expression vector encoding an N-terminal 6-histidine tag and TEV cleavage sites and subsequently expressed in BL21 (DE3) cells.…”

Section: Methodsmentioning

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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Section: Methodsmentioning

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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“…While ubiquitination of H-Ras promotes endosomal localization, 25,26 monoubiquitination at Lys 147 in K-Ras upregulates K-Ras activity by impairing GAP-mediated GTP hydrolysis. 27,28 Moreover, a subset of Rho GTPases is ubiquitinated. Of these, polyubiquitination of RhoA, Rac1, and Cdc42 result in their deactivation and degradation.…”

Section: Mutation Of Cysmentioning

confidence: 99%

Rho GTPases, oxidation, and cell redox control

et al. 2014

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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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Site-specific monoubiquitination activates Ras by impeding GTPase-activating protein function

Cited by 82 publications

References 44 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

Rho GTPases, oxidation, and cell redox control

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

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