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. 2020 Oct 15;10(1):17447.
doi: 10.1038/s41598-020-74463-5.

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

Chih-Chieh Chen  1   2 , Chia-Yi Hsu  3 , Hsiao-Yun Lin  3 , Hong-Qi Zeng  1 , Kuang-Hung Cheng  4 , Chia-Wei Wu  5 , Eing-Mei Tsai  6   7 , Tsung-Hua Hsieh  8
Affiliations

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

Chih-Chieh Chen et al. Sci Rep. .

Abstract

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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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Modeled structures of KRAS and the KRAS/GEF complex. (A,B) Both KRAS (A) and the KRAS/GEF complex (B) are shown. The side chains of the residues to be mutated, Gly12 and Lys104, are represented by yellow spheres. GDP and Mg2+ are shown as a ball-and-stick model and a sphere, respectively. The figures were generated by PyMOL.
Figure 2
Figure 2
Analysis of the structural change of the WT and mutant KRAS/GEF complex in conventional MD simulations. (A) RMSD plot of the WT and mutant KRAS/GEF complex. (B) Schematic illustrating calculation of the dihedral angle. The conformational change of the α2- and α3-helices was detected by measuring the dihedral angle of Cα in Met67, Thr74, Lys104 and Thr87. (C) The dihedral angle of the α2- and α3-helices as a function of time. (D) The dihedral angle distributions in the last 30 ns of the simulation for the α2- and α3-helices are colored blue, red, green and purple for the WT, G12D, G12D + K104A and G12D + K104Q KRAS/GEF complexes, respectively. Dashed lines are mean values. The figures were generated by MATLAB R2015b and PyMOL.
Figure 3
Figure 3
Analysis of atomic fluctuations. (A–D) KRAS/GEF complex structures of (A) WT and (B) G12D, (C) G12D/K104A and (D) G12D/K104Q mutant KRAS are shown as cartoon putty representations; blue represents the lowest and red the highest B-factor value. In addition, the size of the tube reflects the value of the B-factor, whereby the larger is the B-factor, the thicker is the tube. The figures were generated by PyMOL.
Figure 4
Figure 4
Analysis of KRAS/GEF interactions. (A) Two branches of a thermodynamic cycle are placed in one simulation box. The different boxes in the scheme are indicated by the green (λ=0) and blue (λ=1) shading. The free energy estimate corresponds to a double free energy difference: ΔΔG = ΔG1 – ΔG2. The figure was generated by Microsoft PowerPoint 2016. (B) Interactions of KRAS with GEF and RASA1. KRAS was detected by western blot analysis after immunoprecipitation of endogenous GEF and RASA1 in H1299 cells after transfection with KRSWT, KRASG12D, KRASG12D/K104A or KRASG12D/K104Q plasmids. (C–F) Relative levels of GST-Raf1-RBD, RAS, Ac-lysine, p-AKT and AKT detected by western blotting after overexpression of KRASG12D, KRASG12D/K104A or KRASG12D/K104Q plasmids in (C,E) H1299 (KRASWT) and (D,F) MCAS (KRASMT) cancer cell lines. β-Actin was used as a loading control for cell lysates. The full-length and multiple exposures blots are presented in Supplementary Information.
Figure 5
Figure 5
Cellular function of K104 KRASG12D in lung and ovarian cancer. The (A,C,E) H1299 lung cancer cell line and (B,D,F) MCAS ovarian cancer cell line were transfected with KRASWT (1 μg), KRASG12D (1 μg), KRASG12D/K104A (1 μg) or KRASG12D/K104Q (1 μg). After 24 h, (A,B) cell growth was analyzed by CCK-8 assays, (C,D) invasion ability was analyzed with an invasion chamber, and (E,F) migration ability was analyzed by a wound-healing assays (E,F). Data are the mean ± SD from three independent experiments. *P < 0.05 vs. untreated control; two-tailed Student’s t-test. Scale bar = 200 μm.
Figure 6
Figure 6
KRASG12DK104Q inhibits NPIPA2, DUSP1 and IL6 expression compared to KRASG12D and KRASG12D/K104A. (A) H1299 and MCAS cells were transfected with a KRASWT or KRASG12D, KRASG12D/K104A or KRASG12D/K104Q plasmid. We analyzed global gene expression profiles using human oligonucleotide DNA microarrays with three independent RNA samples at 48 h posttransfection. The intensity of each spot was analyzed by GenePix 4.1 software (Molecular Devices). (B) Three identified genes, NPIPA2, DUSP1 and IL6, were upregulated in the presence of KRASG12D and KRASG12D/K104A and downregulated in the presence of KRASG12D/K104Q compared with expression in the control group. (C,D) NPIPA2, DUSP1 and IL6 expression was detected by RT-PCR and after transfection of the mutant forms of KRAS. Data are the mean ± SD from three independent experiments. *P < 0.05 vs. untreated control; two-tailed Student’s t-test.
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