Duplication of Glu37 in the switch I region of HRAS impairs effector/GAP binding and underlies Costello syndrome by promoting enhanced growth factor-dependent MAPK and AKT activation.
Costello syndrome (CS) is a developmental disorder characterized by postnatal reduced growth, facial dysmorphism, cardiac defects, mental retardation and skin and musculo-skeletal defects. CS is caused by HRAS germline mutations. In the majority of cases, mutations affect Gly(12) and Gly(13) and are associated with a relatively homogeneous phenotype. The same amino acid substitutions are well known as somatic mutations in human tumors and promote constitutive HRAS activation by impairing its GTPase activity. In a small number of cases with mild phenotype, a second class of substitutions involving codons 117 and 146 and affecting GTP/GDP binding has been described. Here, we report on the identification and functional characterization of two different three-nucleotide duplications resulting in a duplication of glutamate 37 (p.E37dup) associated with a homogeneous phenotype reminiscent of CS. Ectopic expression of HRAS(E37dup) in COS-7 cells resulted in enhanced growth factor-dependent stimulation of the MEK-ERK and phosphoinositide 3-kinase (PI3K)-AKT signaling pathways. Recombinant HRAS(E37dup) was characterized by slightly increased GTP/GDP dissociation, lower intrinsic GTPase activity and complete resistance to neurofibromin 1 GTPase-activating protein (GAP) stimulation due to dramatically reduced binding. Co-precipitation of GTP-bound HRAS(E37dup) by various effector proteins, however, was inefficient because of drastically diminished binding affinities. Thus, although HRAS(E37dup) is predominantly present in the active, GTP-bound state, it promotes only a weak hyperactivation of downstream signaling pathways. These findings provide evidence that the mildly enhanced signal flux through the MAPK and PI3K-AKT cascades promoted by these disease-causing germline HRAS alleles results from a balancing effect between a profound GAP insensitivity and inefficient binding to effector proteins.
Gremer
Lothar
L
Institute of Biochemistry and Molecular Biology II, Heinrich Heine University Medical Center, 40225 Düsseldorf, Germany.
De Luca
Alessandro
A
Merbitz-Zahradnik
Torsten
T
Dallapiccola
Bruno
B
Morlot
Susanne
S
Tartaglia
Marco
M
Kutsche
Kerstin
K
Ahmadian
Mohammad Reza
MR
Rosenberger
Georg
G
D000483:Alleles; D000818:Animals; D019556:COS Cells; D002648:Child; D002675:Child, Preschool; D002522:Chlorocebus aethiops; D056685:Costello Syndrome / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D019066:Facies; D011905:Genes, ras; D018698:Glutamic Acid / Q000235:genetics*; D006801:Humans; D020935:MAP Kinase Signaling System / Q000502:physiology*; D009154:Mutation; D025542:Neurofibromin 1 / Q000378:metabolism; D010641:Phenotype; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction
D025542:Neurofibromin 1; D018698:Glutamic Acid; D051057:Proto-Oncogene Proteins c-akt; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)