Insights into K-Ras 4B regulation by post-translational lysine acetylation

Knyphausen, Philipp; Lang, Franziska; Baldus, Linda; Extra, Antje; Lammers, Michael

doi:10.1515/hsz-2016-0118

Cited by 31 publications

(43 citation statements)

References 77 publications

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“…Although K104Q has been employed as an acetylation mimetic, this has yet to be truly validated. In fact, Lys 104 acetylation has recently been reported to retain SOS activity (10). Computational analysis of the Ku protein revealed that acetylation of the Ku may not alter DNA interaction, yet a K-to-Q mutation decreased the binding compared with the WT protein (31).…”

Section: Discussionmentioning

confidence: 99%

“…104 Proteins-His 6 -WT KRAS (residues 1-169, containing a 12-amino acid N-terminal non-cleavable His 6 tag) and K104Q were expressed in LB medium as His 6 -tagged fusion proteins (pRSF-Duet, Merck Biosciences) in E. coli BL21 (DE3) cells as described (10). The E. coli culture was grown to an A 600 of 0.6 (37°C; 160 rpm), and protein expression was subsequently induced by the addition of 300 M IPTG and further incubated overnight for 16 h (18°C, 160 rpm).…”

Section: Expression and Purification Of His 6 -Wt Kras K104q And Acmentioning

confidence: 99%

“…In contrast, lysine 104 acetylation was reported to down-regulate KRAS G12V-driven effector signaling and growth transformation in NIH 3T3 cells (8,9). Whereas knockdown of two deacetylases, HDAC6 and SIRT2, reduced the viability of NIH 3T3 cells expressing the oncogenic KRAS G12V mutant (9), recent findings indicate that Ac-Lys 104 is not a direct substrate for HDAC6 and SIRT2 under the conditions tested (10). A KRAS K104Q variant was used as an acetylation mimetic to evaluate how acetylation alters KRAS signaling.…”

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confidence: 90%

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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: Discussionmentioning

confidence: 99%

Section: Expression and Purification Of His 6 -Wt Kras K104q And Acmentioning

confidence: 99%

mentioning

confidence: 90%

See 1 more Smart Citation

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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“…Hence, elucidation of the RAS signal transduction requires not only RAS-effector interactions but also additional structures and interplay of multiprotein complexes [25]. Another critical aspect is sorting/trafficking of the isoforms [83,84] that has recently been shown to be highly specific for the respective RAS proteins and dependents on specific posttranslational modifications, including prenylation and acylation [85,86], phosphorylation [87,88], ubiquitination [89,90,91,92] and acetylation [93,94,95]. Similar characteristics have been reported for the RRAS isoforms, including protein-protein interaction required for subcellular localization, e.g., at focal adhesion or recycling endosomes,[96,97], and posttranslational modifications [98,99,100].…”

Section: Discussionmentioning

confidence: 99%

The RAS-Effector Interface: Isoform-Specific Differences in the Effector Binding Regions

Nakhaeizadeh¹,

Amin²,

Nakhaei‐Rad³

et al. 2016

PLoS ONE

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RAS effectors specifically interact with the GTP-bound form of RAS in response to extracellular signals and link them to downstream signaling pathways. The molecular nature of effector interaction by RAS is well-studied but yet still incompletely understood in a comprehensive and systematic way. Here, structure-function relationships in the interaction between different RAS proteins and various effectors were investigated in detail by combining our in vitro data with in silico data. Equilibrium dissociation constants were determined for the binding of HRAS, KRAS, NRAS, RRAS1 and RRAS2 to both the RAS binding (RB) domain of CRAF and PI3Kα, and the RAS association (RA) domain of RASSF5, RALGDS and PLCε, respectively, using fluorescence polarization. An interaction matrix, constructed on the basis of available crystal structures, allowed identification of hotspots as critical determinants for RAS-effector interaction. New insights provided by this study are the dissection of the identified hotspots in five distinct regions (R1 to R5) in spite of high sequence variability not only between, but also within, RB/RA domain-containing effectors proteins. Finally, we propose that intermolecular β-sheet interaction in R1 is a central recognition region while R3 may determine specific contacts of RAS versus RRAS isoforms with effectors.

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“…For immunoprecipitation experiments, 2 mg of cell lysate was incubated with specific antibodies and immobilized on protein A/G PLUS-agarose beads (Santa Cruz Biotechnology). Proteins were separated by SDS-PAGE, transferred to a nitrocellulose membrane and blotted with specific antibodies, including those against RAS (1:300, Thermo Fisher Scientific, #16117) 25 , GEF H1 (1:500, Abcam, ab155785) 26 , RASA1 (1:500, Abcam, ab2922) 27 , Ac-lysine (1:500, Abcam, ab21623) 28,29 , phospho-AKT (Ser-473, 1:500, Cell Signaling, #4051) 30 , AKT (1:500, Cell Signaling, #2920) 31 and β-actin (1;1000, Thermo Fisher Scientific, #AM4302) 32 , for western blotting.…”

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confidence: 99%

KRAS K104 modification affects the KRASG12D-GEF interaction and mediates cell growth and motility

Chen

Hsu

Lin

et al. 2020

Sci Rep

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Mutant RAS genes play an important role in regulating tumors through lysine residue 104 to impair GEF-induced nucleotide exchange, but the regulatory role of KRAS K104 modification on the KRASG12D mutant remains unclear. Therefore, we simulated the acetylation site on the KRASG12D three-dimensional protein structure, including KRASG12D, KRASG12D/K104A and KRASG12D/K104Q, and determined their trajectories and binding free energy with GEF. KRASG12D/K104Q induced structural changes in the α2- and α3-helices, promoted KRAS instability and hampered GEF binding (ΔΔG = 6.14 kJ/mol). We found decreased binding to the Raf1 RBD by KRASG12D/K104Q and reduced cell growth, invasion and migration. Based on whole-genome cDNA microarray analysis, KRASG12D/K104Q decreased expression of NPIPA2, DUSP1 and IL6 in lung and ovarian cancer cells. This study reports computational and experimental analyses of Lys104 of KRASG12D and GEF, and the findings provide a target for exploration for future treatment.

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Insights into K-Ras 4B regulation by post-translational lysine acetylation

Cited by 31 publications

References 77 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

The RAS-Effector Interface: Isoform-Specific Differences in the Effector Binding Regions

KRAS K104 modification affects the KRASG12D-GEF interaction and mediates cell growth and motility

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