Popov IK et al. (2019), A YWHAZ Variant Associated With Cardiofaciocuta...

XB-ART-55907

Front Physiol 2019 Jan 01;10:388. doi: 10.3389/fphys.2019.00388.

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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 .

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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.

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Species referenced: Xenopus laevis
Genes referenced: braf csnk1a1 fgfr1 kras map2k1 map2k4 mapk1 ptpn11 raf1 sos1 tbxt ywhaz
GO keywords: gastrulation

???displayArticle.disOnts??? Costello syndrome [+]
Phenotypes: Xla + Hsa.YWHAZ (Fig 2C middle panel) [+]

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	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 back”). 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

References [+] :

Aitken, 14-3-3 proteins: a historic overview. 2006, Pubmed