Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
Front Physiol
2019 Jan 01;10:388. doi: 10.3389/fphys.2019.00388.
Show Gene links
Show Anatomy links
A YWHAZ Variant Associated With Cardiofaciocutaneous Syndrome Activates the RAF-ERK Pathway.
Popov IK, Hiatt SM, Whalen S, Keren B, Ruivenkamp C, van Haeringen A, Chen MJ, Cooper GM, Korf BR, Chang C.
???displayArticle.abstract???
Cardiofaciocutaneous (CFC) syndrome is a genetic disorder characterized by distinctive facial features, congenital heart defects, and skin abnormalities. Several germline gain-of-function mutations in the RAS/RAF/MEK/ERK pathway are associated with the disease, including KRAS, BRAF, MEK1, and MEK2. CFC syndrome thus belongs to a group of disorders known as RASopathies, which are all caused by pathogenic mutations in various genes encoding components of the RAS pathway. We recently identified novel variants in YWHAZ, a 14-3-3 family member, in individuals with a phenotype consistent with CFC that may potentially be deleterious and disease-causing. In the current study, we take advantage of the vertebrate model Xenopus laevis to analyze the functional consequence of a particular YWHAZ variant, S230W, and investigate the molecular mechanisms underlying its activity. We show that compared with wild type YWHAZ, the S230W variant induces severe embryonic defects when ectopically expressed in early Xenopus embryos. The S230W variant also rescues the defects induced by a dominant negative FGF receptor more efficiently and enhances Raf-stimulated Erk phosphorylation to a higher level than wild type YWHAZ. Although neither YWHAZ nor the variant promotes membrane recruitment of Raf proteins, the variant binds to more Raf and escapes phosphorylation by casein kinase 1a. Our data provide strong support to the hypothesis that the S230W variant of YWHAZ is a gain-of-function mutation in the RAS-ERK pathway and may underlie a CFC phenotype.
FIGURE 1. Facial features of probands with variations in YWHAZ. (A) Proband 1. (B) Proband 2. (C) Proband 3 (photo taken at the age of 13 and a half years old). (D) Proband 4 (photo taken at the age of 4 years and 8 months old). (E) Proband 5 (photo taken at the age of 7 years and 5 months old). Written informed consent to publish pictures was obtained from all families.
FIGURE 2. YWHAZ(S230W) induces more severe embryonic defects than wild type YWHAZ. (A) Ectopic expression of YWHAZ(S230W) in the animal region induces darkly pigmented cells at the gastrula stages. Arrows point to the darkly pigmented cell clusters. (B) Clusters of darkly pigmented cells induced by ectodermal expression of YWHAZ(S230W) persist to the neurula stages. Arrows point to the darkly pigmented cell clusters. (C) Overexpression of wild type YWHAZ leads to defects in the head structure and bent body axis in the resulting tadpoles. Expression of the YWHAZ(S230W) variant induces more severe defects than YWHAZ, with the tadpoles displaying a significantly shortened and severely bent body axis in addition to stronger defects in the head structure. The experiment has been repeated four times, with 10 to 18 treated embryos in each experiment. In all the experiments, the length of the embryos was significantly shorter in the YWHAZ(S230W)-expressing group. RNA doses are 2–4 ng.
FIGURE 3. YWHAZ(S230W) rescues embryonic defects induced by DN-FGFR1 more efficiently than YWHAZ. (A) Expression of DN-FGFR1 (25 pg or 50 pg) in the dorsal marginal zone of early embryos results in gastrulation defects, characterized by reduced body length and exposed mesendoderm (“open backâ€). Co-expression of either YWHAZ or YWHAZ(S230W) (250 pg) with DN-FGFR1 leads to phenotypic rescue, but the S230W variant is more efficient in restoring body length and embryo morphology. The scatter plot of the body length of the embryos showed that the S230W variant rescued the defects more efficiently than YWHAZ. Student’s t-tests were performed in a pairwise fashion and the results revealed significant differences in the samples we compared. This experiment has been repeated four times, with 12 to 18 treated embryos in each experiment. Since the embryos were collected at slightly different stages and the body length was thus different, scatter plot was made for the embryos for each experiment separately. In all the experiments, there was a significant difference between YWHAZ and YWHAZ(S230W) in phenotype rescue. (B) DN-FGFR1 interferes with the expression of the pan-mesodermal marker brachyury, and YWHAZ(S230W) rescues marker expression to a greater extent compared to wild type YWHAZ. The experiment was repeated 3 times. For control embryos, 1/44 embryos showed a gap in the brachyury ring, and 33/38 embryos injected with DN-FGFR1 RNA had a big gap in the brachyury domain. Expression of YWHAZ and YWHAZ(S230W) led to partial rescue of brachyury expression, resulting in similar brachyury gap in 24/38 and 13/38 embryos, respectively. These numbers are indicated in the panel. (C) Ectopic expression of YWHAZ weakly expands, whereas ectopic expression of YWHAZ(S230W) strongly expands, the domain of brachyury in gastrulating Xenopus embryos. The experiment has been repeated 3 times. The expansion of brachyury domain was observed in 16/30 embryos expressing YWHAZ and 24/30 embryos expressing YWHAZ(S230W). The doses of RNAs used in this panel are 1 to 2 ng. The red color in panels B and C are staining of beta-galactosidase-expressing cells with the chemical red-Gal to mark the injected cells.
FIGURE 4. YWHAZ(S230W) strongly stimulates Raf-dependent Erk phosphorylation when compared with wild type YWHAZ. (A) Co-expression of Craf (250 pg) and GFP-Erk2 (250 pg) with wild type YWHAZ, the S230W variant, or the T232A mutant (250 pg) shows that only the S230W variant strongly enhances Craf-stimulated Erk phosphorylation. Quantification of the Western blot was performed using NIH ImageJ software and the relative ratio of phosphorylated GFP-Erk2 (dpGFP-Erks) over total GFP-Erk2 is shown in the bar graph. (B) Co-expression of Craf (125 pg), GFP-Erk2 (125 pg) and an increasing amount of HA-YWHAZ or HA-YWHAZ(S230W) (125, 250, and 500 pg) shows that the S230W variant stimulates Erk phosphorylation significantly more than YWHAZ at all doses used. The bottom bar graph displays the results of three independent experiments, with the average of relative Erk phosphorylation level (dpGFP-Erk2/total GFP-Erk2) and the standard deviation plotted on the graph. The p-values from Student’s t-test for each dose of YWHAZ/S230W are shown in the graph.
FIGURE 5. YWHAZ and YWHAZ(S230W) do not promote membrane recruitment of Raf. (A) Craf-GFP is localized both at the plasma membrane and in the cytosol in Xenopus ectodermal cells. Co-expression with YWHAZ or YWHAZ(S230W) does not significantly change the membrane fraction of the protein. The surface view reveals a population of Craf at the cell-cell junction in the presence of YWHAZ or the S230W variant in addition to the widespread Craf underneath the cell surface. Cell morphological changes are observed only in cells expressing the S230W variant and manifested as clusters of smaller cells displaying apical constriction-like morphology from the surface view (arrows). The side view shows intense Craf clusters at the juxtamembrane locations in cells expressing YWHAZ or YWHAZ(S230W). (B) Unlike wild type YWHAZ or its variant, FGFR1 recruits Craf to the plasma membrane in ectodermal cells. The doses of the RNAs used are: Craf-GFP, 250 pg, YWHAZ/YWHAZ(S230W), 2 ng, FGFR1, 1 ng.
FIGURE 6. YWHAZ(S230W) binds to more Raf proteins than YWHAZ. Binding of HA-tagged YWHAZ(S230W) to either Craf (panel A) or Braf (panel B) is increased compared to that of HA-tagged YWHAZ. The doses of the RNAs used are: Craf-GFP, 250 pg, Braf-Flag, 125 pg, HA-YWHAZ/HA-YWHAZ(S230W)/HA-YWHAZ(T232A), 250 pg.
FIGURE 7. YWHAZ(S230W) cannot be phosphorylated by casein kinase 1a (CK1a). The S230W variant, like the phospho-null T232A mutant of YWHAZ, cannot be phosphorylated by CK1a. The doses of RNAs used are: HA-YWHAZ/HA-YWHAZ(S230W)/HA-YWHAZ(T232A), 1 ng; Flag-CK1a, 500 pg.
Supplementary Image 1. YWHAZ Variant Associated With Cardiofaciocutaneous Syndrome Activates the RAF-ERK Pathway
Supplementary Image 2. YWHAZ Variant Associated With Cardiofaciocutaneous Syndrome Activates the RAF-ERK Pathway
Aitken,
14-3-3 proteins: a historic overview.
2006, Pubmed
Aitken,
14-3-3 proteins: a historic overview.
2006,
Pubmed Amaya,
FGF signalling in the early specification of mesoderm in Xenopus.
1993,
Pubmed
,
Xenbase Amaya,
Expression of a dominant negative mutant of the FGF receptor disrupts mesoderm formation in Xenopus embryos.
1991,
Pubmed
,
Xenbase Anastasaki,
Kinase-activating and kinase-impaired cardio-facio-cutaneous syndrome alleles have activity during zebrafish development and are sensitive to small molecule inhibitors.
2009,
Pubmed Aoki,
The RAS/MAPK syndromes: novel roles of the RAS pathway in human genetic disorders.
2008,
Pubmed Bowling,
Genomic diagnosis for children with intellectual disability and/or developmental delay.
2017,
Pubmed Brennan,
A Raf-induced allosteric transition of KSR stimulates phosphorylation of MEK.
2011,
Pubmed Bunney,
Fusicoccin signaling reveals 14-3-3 protein function as a novel step in left-right patterning during amphibian embryogenesis.
2003,
Pubmed
,
Xenbase Bustelo,
RAS GTPase-dependent pathways in developmental diseases: old guys, new lads, and current challenges.
2018,
Pubmed Casar,
Essential role of ERK dimers in the activation of cytoplasmic but not nuclear substrates by ERK-scaffold complexes.
2008,
Pubmed Dard,
RAS signalling in energy metabolism and rare human diseases.
2018,
Pubmed Dubois,
14-3-3 is phosphorylated by casein kinase I on residue 233. Phosphorylation at this site in vivo regulates Raf/14-3-3 interaction.
1997,
Pubmed Fantl,
Activation of Raf-1 by 14-3-3 proteins.
1994,
Pubmed
,
Xenbase Firth,
DECIPHER: Database of Chromosomal Imbalance and Phenotype in Humans Using Ensembl Resources.
2009,
Pubmed Fischer,
Regulation of RAF activity by 14-3-3 proteins: RAF kinases associate functionally with both homo- and heterodimeric forms of 14-3-3 proteins.
2009,
Pubmed Freeman,
Effects of Raf dimerization and its inhibition on normal and disease-associated Raf signaling.
2013,
Pubmed Garnett,
Wild-type and mutant B-RAF activate C-RAF through distinct mechanisms involving heterodimerization.
2005,
Pubmed Gotoh,
Involvement of the MAP kinase cascade in Xenopus mesoderm induction.
1995,
Pubmed
,
Xenbase Harland,
In situ hybridization: an improved whole-mount method for Xenopus embryos.
1991,
Pubmed
,
Xenbase Heidorn,
Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
2010,
Pubmed Hekman,
Dynamic changes in C-Raf phosphorylation and 14-3-3 protein binding in response to growth factor stimulation: differential roles of 14-3-3 protein binding sites.
2004,
Pubmed James-Zorn,
Xenbase: Core features, data acquisition, and data processing.
2015,
Pubmed
,
Xenbase Karimi,
Xenbase: a genomic, epigenomic and transcriptomic model organism database.
2018,
Pubmed
,
Xenbase Khokhlatchev,
Phosphorylation of the MAP kinase ERK2 promotes its homodimerization and nuclear translocation.
1998,
Pubmed Kimelman,
The presence of fibroblast growth factor in the frog egg: its role as a natural mesoderm inducer.
1988,
Pubmed
,
Xenbase LaBonne,
Localization of MAP kinase activity in early Xenopus embryos: implications for endogenous FGF signaling.
1997,
Pubmed
,
Xenbase LaBonne,
Mesoderm induction by activin requires FGF-mediated intracellular signals.
1994,
Pubmed
,
Xenbase LaBonne,
Role of MAP kinase in mesoderm induction and axial patterning during Xenopus development.
1995,
Pubmed
,
Xenbase Lau,
Differential role of 14-3-3 family members in Xenopus development.
2006,
Pubmed
,
Xenbase Lavoie,
MEK drives BRAF activation through allosteric control of KSR proteins.
2018,
Pubmed Lavoie,
Regulation of RAF protein kinases in ERK signalling.
2015,
Pubmed Lek,
Analysis of protein-coding genetic variation in 60,706 humans.
2016,
Pubmed Light,
14-3-3 antagonizes Ras-mediated Raf-1 recruitment to the plasma membrane to maintain signaling fidelity.
2002,
Pubmed Liu,
Crystal structure of the zeta isoform of the 14-3-3 protein.
1995,
Pubmed
,
Xenbase MacNicol,
Raf-1 kinase is essential for early Xenopus development and mediates the induction of mesoderm by FGF.
1993,
Pubmed
,
Xenbase MacNicol,
Disruption of the 14-3-3 binding site within the B-Raf kinase domain uncouples catalytic activity from PC12 cell differentiation.
2000,
Pubmed
,
Xenbase Morrison,
The 14-3-3 proteins: integrators of diverse signaling cues that impact cell fate and cancer development.
2009,
Pubmed Müller,
C-TAK1 regulates Ras signaling by phosphorylating the MAPK scaffold, KSR1.
2001,
Pubmed Muslin,
Interaction of 14-3-3 with signaling proteins is mediated by the recognition of phosphoserine.
1996,
Pubmed
,
Xenbase Muslin,
Differential functions of 14-3-3 isoforms in vertebrate development.
2005,
Pubmed Nie,
PI3K and Erk MAPK mediate ErbB signaling in Xenopus gastrulation.
2007,
Pubmed
,
Xenbase Obsil,
Structural basis of 14-3-3 protein functions.
2011,
Pubmed Obsilova,
14-3-3zeta C-terminal stretch changes its conformation upon ligand binding and phosphorylation at Thr232.
2004,
Pubmed Pearl,
Development of Xenopus resource centers: the National Xenopus Resource and the European Xenopus Resource Center.
2012,
Pubmed
,
Xenbase Richards,
Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology.
2015,
Pubmed Roberts,
The cardiofaciocutaneous syndrome.
2006,
Pubmed Rommel,
Activated Ras displaces 14-3-3 protein from the amino terminus of c-Raf-1.
1996,
Pubmed Roy,
KSR is a scaffold required for activation of the ERK/MAPK module.
2002,
Pubmed Sawyer,
Apical constriction: a cell shape change that can drive morphogenesis.
2010,
Pubmed
,
Xenbase Schohl,
Beta-catenin, MAPK and Smad signaling during early Xenopus development.
2002,
Pubmed
,
Xenbase Simanshu,
RAS Proteins and Their Regulators in Human Disease.
2017,
Pubmed Sluchanko,
Association of Multiple Phosphorylated Proteins with the 14-3-3 Regulatory Hubs: Problems and Perspectives.
2018,
Pubmed Smith,
Expression of a Xenopus homolog of Brachyury (T) is an immediate-early response to mesoderm induction.
1991,
Pubmed
,
Xenbase Sobreira,
GeneMatcher: a matching tool for connecting investigators with an interest in the same gene.
2015,
Pubmed Therrien,
KSR modulates signal propagation within the MAPK cascade.
1996,
Pubmed
,
Xenbase Tien,
Snail2/Slug cooperates with Polycomb repressive complex 2 (PRC2) to regulate neural crest development.
2015,
Pubmed
,
Xenbase Truong,
Role of the 14-3-3 C-terminal loop in ligand interaction.
2002,
Pubmed Tzivion,
A dimeric 14-3-3 protein is an essential cofactor for Raf kinase activity.
1998,
Pubmed Umbhauer,
Mesoderm induction in Xenopus caused by activation of MAP kinase.
1995,
Pubmed
,
Xenbase Weber,
Active Ras induces heterodimerization of cRaf and BRaf.
2001,
Pubmed Wu,
Role of 14-3-3 proteins in early Xenopus development.
2002,
Pubmed
,
Xenbase Yaffe,
The structural basis for 14-3-3:phosphopeptide binding specificity.
1997,
Pubmed
