A KRAS GTPase K104Q Mutant Retains Downstream Signaling by Offsetting Defects in Regulation.
The 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 itsin vitroand cell-based 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 allRasgenes. 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.
From the Department of Biochemistry and Biophysics.
Guowei
0000-0003-2669-7160
G
Yin
From the Department of Biochemistry and Biophysics.|Department of Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy.
Samantha
S
Kistler
Department of Pharmacology, and.|Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27699.
Samuel D
SD
George
the Institute for Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging-associated Diseases (CECAD), Joseph-Stelzmann-Strasse 26, University of Cologne, 50931 Cologne, Germany, and.
Nora
N
Kuhlmann
the NCI RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, Maryland 21702.
Leslie
L
Garvey
From the Department of Biochemistry and Biophysics.|Department of Pharmacology, and.|Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27699.
Minh
M
Huynh
the NCI RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, Maryland 21702.
Rachel K
RK
Bagni
the Institute for Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging-associated Diseases (CECAD), Joseph-Stelzmann-Strasse 26, University of Cologne, 50931 Cologne, Germany, and.
Michael
M
Lammers
Department of Pharmacology, and.|Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27699.
Channing J
CJ
Der
From the Department of Biochemistry and Biophysics, campbesl@med.unc.edu.|Department of Pharmacology, and.|Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27699.
Sharon L
SL
Campbell
D000818:Animals; D002478:Cells, Cultured; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D051379:Mice; D008958:Models, Molecular; D020125:Mutation, Missense; D041681:NIH 3T3 Cells; D017354:Point Mutation; D011487:Protein Conformation; D055550:Protein Stability; D016283:Proto-Oncogene Proteins p21(ras) / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015398:Signal Transduction
D020662:Guanine Nucleotide Exchange Factors; C117307:KRAS protein, human; D006160:Guanosine Triphosphate; C495971:Hras protein, mouse; D016283:Proto-Oncogene Proteins p21(ras)
GTPase-activating protein (GAP); Ras protein; guanine nucleotide exchange factor (GEF); nuclear magnetic resonance (NMR); post-translational modification; protein acetylation; signaling; tumorigenesis