CRAF Autophosphorylation of Serine 621 Is Required to Prevent Its Proteasome-Mediated Degradation

Noble, Catherine; Mercer, Kathryn; Hussain, Jahan; Carragher, Linda A. S.; Giblett, Susan; Hayward, Robert; Patterson, Cam; Marais, Richard; Pritchard, Catrin

doi:10.1016/j.molcel.2008.08.026

Cited by 82 publications

(110 citation statements)

References 46 publications

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“…Notably, in D486N/ϩ MEFs, Raf1 protein levels were only ϳ60% of WT levels, a finding consistent with previous work showing that Raf1 kinase activity is required to prevent the ubiquitin-mediated proteolysis of Raf1 (27). Nevertheless, D486N/ϩ MEFs showed sustained Mek and Erk activation in response to EGF (Fig.…”

Section: Generation Of Raf1supporting

confidence: 91%

Increased BRAF Heterodimerization Is the Common Pathogenic Mechanism for Noonan Syndrome-Associated RAF1 Mutants

Yin

Simpson

et al. 2012

Molecular and Cellular Biology

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Noonan syndrome (NS) is a relatively common autosomal dominant disorder characterized by congenital heart defects, short stature, and facial dysmorphia. NS is caused by germ line mutations in several components of the RAS-RAF-MEK-extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway, including both kinase-activating and kinaseimpaired alleles of RAF1 (ϳ3 to 5%), which encodes a serine-threonine kinase for MEK1/2. To investigate how kinase-impaired RAF1 mutants cause NS, we generated knock-in mice expressing Raf1 D486N . Raf1 D486N/؉ (here D486N/؉) female mice exhibited a mild growth defect. Male and female D486N/D486N mice developed concentric cardiac hypertrophy and incompletely penetrant, but severe, growth defects. Remarkably, Mek/Erk activation was enhanced in Raf1 D486N -expressing cells compared with controls. RAF1 D486N , as well as other kinase-impaired RAF1 mutants, showed increased heterodimerization with BRAF, which was necessary and sufficient to promote increased MEK/ERK activation. Furthermore, kinase-activating RAF1 mutants also required heterodimerization to enhance MEK/ERK activation. Our results suggest that an increased heterodimerization ability is the common pathogenic mechanism for NS-associated RAF1 mutations. N oonan syndrome (NS) is a relatively common (1 in 1,000 to 2,500 live births) autosomal dominant disorder (28,30,45), characterized by short stature, craniofacial dysmorphia, a wide spectrum of congenital cardiac anomalies, and an increased risk of hematopoietic malignancy. Although NS is genetically heterogeneous (1, 4, 49), all known cases are caused by germ line mutations in conserved components of the canonical RAS-RAF-MEK-extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) (here RAS/ERK) cascade, a key regulator of cell proliferation, differentiation, and survival (13, 24). Mutations in PTPN11, which encodes the protein tyrosine phosphatase SHP2, account for approximately one-half of NS cases (46). Other known NS genes include SOS1 (ϳ10%) (38, 47), RAF1 (3 to 5%) (31, 36), KRAS (Ͻ2%) (41, 56), NRAS (9), and SHOC2 (10) (Ͻ1 to 2%). Mutations in some of these genes, as well as in genes encoding other RAS/ERK pathway components, also cause phenotypically related disorders, such as neurofibromatosis type 1 (NF1), Costello syndrome, cardiofacio-cutaneous (CFC) syndrome, and LEOPARD syndrome (named for the major features of this disorder, including multiple lentigines, electrocardiographic conduction abnormalities, ocular hypertelorism, pulmonic stenosis, abnormal genitalia, retardation of growth, and sensorneural deafness); together with NS, these syndromes are now termed "RASopathies" (49). How mutations in the same signaling pathway cause similar yet clearly distinct phenotypes remains unclear. Consequently, a detailed understanding of RASopathy pathogenesis should yield new insights into RAS/ERK pathway regulation.RAF family serine-threonine kinases (22,25,53) function as key RAS effectors, phosphorylating and a...

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Section: Generation Of Raf1supporting

confidence: 91%

Increased BRAF Heterodimerization Is the Common Pathogenic Mechanism for Noonan Syndrome-Associated RAF1 Mutants

Yin

Simpson

et al. 2012

Molecular and Cellular Biology

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“…MEFs were infected with AdCre or Adbgal by addition of ;1 3 10 8 plaque-forming units (pfu) directly to the culture medium. For growth assays, cells were plated at a density of 3 3 10 4 in triplicate and recounted every 2 d for 8 d. For immortalization assays, MEFs were plated at 3 3 10 5 cells per 6-cm plate in triplicate, counted, and replated every 3 d. For Braf and Craf siRNA, ON-TARGETplus SMARTpool siRNAs (Dharmacon) were used and transfected using Lipofectamine 2000 as previously described (Noble et al 2008). For inhibitor treatments, MEFs at ;80% confluency were treated with 10 mM U0126, 1 mM PD184352, 0.3 mM PLX4720, or 1 mM SB590885 for 4 h or a volume of DMSO equivalent as the carrier control.…”

Section: Cells and Treatmentsmentioning

confidence: 99%

The intermediate-activity^L597VBRAF mutant acts as an epistatic modifier of oncogenic RAS by enhancing signaling through the RAF/MEK/ERK pathway

et al. 2012

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L597V BRAF mutations are acquired somatically in human cancer samples and are frequently coincident with RAS mutations. Germline L597V BRAF mutations are also found in several autosomal dominant developmental conditions known as RASopathies, raising the important question of how the same mutation can contribute to both pathologies. Using a conditional knock-in mouse model, we show that endogenous expression of L597V Braf leads to approximately twofold elevated Braf kinase activity and weak activation of the Mek/Erk pathway. This is associated with induction of RASopathy hallmarks including cardiac abnormalities and facial dysmorphia but is not sufficient for tumor formation. We combined L597V Braf with G12D Kras and found that L597V Braf modified G12D Kras oncogenesis such that fibroblast transformation and lung tumor development were more reminiscent of that driven by the high-activity V600E Braf mutant. Mek/Erk activation levels were comparable with those driven by V600E Braf in the double-mutant cells, and the gene expression signature was more similar to that induced by V600E Braf than G12D Kras. However, unlike V600E Braf, Mek/Erk pathway activation was mediated by both Craf and Braf, and ATP-competitive RAF inhibitors induced paradoxical Mek/ Erk pathway activation. Our data show that weak activation of the Mek/Erk pathway underpins RASopathies, but in cancer, L597V Braf epistatically modifies the transforming effects of driver oncogenes.

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“…Most well-established examples are kinase cascades in which phosphorylation/dephosphorylation events can function as the basis for signal transduction. A number of kinases can be activated by an autoregulatory system called autophosphorylation (1,2). Discovery of the autoacetylation could extend this autoregulatory system to acetyltransferases (3).…”

Section: Introductionmentioning

confidence: 99%

Arrest Defective 1 Autoacetylation Is a Critical Step in Its Ability to Stimulate Cancer Cell Proliferation

Seo

Park

et al. 2010

Cancer Research

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The N-acetyltransferase arrest defective 1 (ARD1) is an important regulator of cell growth and differentiation that has emerged recently as a critical molecule in cancer progression. However, the regulation of the enzymatic and biological activities of human ARD1 (hARD1) in cancer is presently poorly understood. Here, we report that hARD1 undergoes autoacetylation and that this modification is essential for its functional activation. Using liquid chromatography-tandem mass spectrometry and site-directed mutational analyses, we identified K136 residue as an autoacetylation target site. K136R mutation abolished the ability of hARD1 to promote cancer cell growth in vitro and tumor xenograft growth in vivo. Mechanistic investigations revealed that hARD1 autoacetylation stimulated cyclin D1 expression through activation of the transcription factors β-catenin and activator protein-1. Our results show that hARD1 autoacetylation is critical for its activation and its ability to stimulate cancer cell proliferation and tumorigenesis. Cancer Res; 70(11); 4422-32. ©2010 AACR.

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CRAF Autophosphorylation of Serine 621 Is Required to Prevent Its Proteasome-Mediated Degradation

Cited by 82 publications

References 46 publications

Increased BRAF Heterodimerization Is the Common Pathogenic Mechanism for Noonan Syndrome-Associated RAF1 Mutants

Increased BRAF Heterodimerization Is the Common Pathogenic Mechanism for Noonan Syndrome-Associated RAF1 Mutants

The intermediate-activity^L597VBRAF mutant acts as an epistatic modifier of oncogenic RAS by enhancing signaling through the RAF/MEK/ERK pathway

Arrest Defective 1 Autoacetylation Is a Critical Step in Its Ability to Stimulate Cancer Cell Proliferation

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CRAF Autophosphorylation of Serine 621 Is Required to Prevent Its Proteasome-Mediated Degradation

Cited by 82 publications

References 46 publications

Increased BRAF Heterodimerization Is the Common Pathogenic Mechanism for Noonan Syndrome-Associated RAF1 Mutants

Increased BRAF Heterodimerization Is the Common Pathogenic Mechanism for Noonan Syndrome-Associated RAF1 Mutants

The intermediate-activityL597VBRAF mutant acts as an epistatic modifier of oncogenic RAS by enhancing signaling through the RAF/MEK/ERK pathway

Arrest Defective 1 Autoacetylation Is a Critical Step in Its Ability to Stimulate Cancer Cell Proliferation

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The intermediate-activity^L597VBRAF mutant acts as an epistatic modifier of oncogenic RAS by enhancing signaling through the RAF/MEK/ERK pathway