BRAF activates PAX3 to control muscle precursor cell migration during forelimb muscle development

Cetinkaya

et al. 2020

IJMS

Self Cite

Fetal and hypertrophic remodeling are hallmarks of cardiac restructuring leading chronically to heart failure. Since the Ras/Raf/MEK/ERK cascade (MAPK) is involved in the development of heart failure, we hypothesized, first, that fetal remodeling is different from hypertrophy and, second, that remodeling of the MAPK occurs. To test our hypothesis, we analyzed models of cultured adult rat cardiomyocytes as well as investigated myocytes in the failing human myocardium by western blot and confocal microscopy. Fetal remodeling was induced through endothelial morphogens and monitored by the reexpression of Acta2, Actn1, and Actb. Serum-induced hypertrophy was determined by increased surface size and protein content of cardiomyocytes. Serum and morphogens caused reprogramming of Ras/Raf/MEK/ERK. In both models H-Ras, N-Ras, Rap2, B- and C-Raf, MEK1/2 as well as ERK1/2 increased while K-Ras was downregulated. Atrophy, MAPK-dependent ischemic resistance, loss of A-Raf, and reexpression of Rap1 and Erk3 highlighted fetal remodeling, while A-Raf accumulation marked hypertrophy. The knock-down of B-Raf by siRNA reduced MAPK activation and fetal reprogramming. In conclusion, we demonstrate that fetal and hypertrophic remodeling are independent processes and involve reprogramming of the MAPK.

Section: Discussionmentioning

confidence: 99%

The MEK/ERK Module Is Reprogrammed in Remodeling Adult Cardiomyocytes

Cetinkaya

et al. 2020

IJMS

Self Cite

“…Following upstream stimulation by ligand-activated receptor tyrosine kinases, BRAF forms a network of interacting kinases including ARAF, CRAF and KSR1/2 that bind to RAS.GTP at the plasma membrane, leading to RAF phosphorylation of MEK1/2, which in turn activates ERK1/2 [52]. Despite this widely-accepted model of BRAF regulation, a number of other subcellular locations have been reported for BRAF [27, 28, 29, 30, 31, 32, 56]. Here, using fluorescence imaging and fractionation of tagged, exogenous proteins expressed in NIH3T3 cells, we provide evidence in support of the location of wild-type and oncogenic forms of full length BRAF in the cytoplasm of the cell.…”

Section: Discussionmentioning

confidence: 99%

“…For example, a portion of BRAF has been detected at spindle poles and kinetochores in mitotic HeLa cells and knockdown of BRAF using siRNA resulted in early exit of cells from mitosis, perturbation of Mps1 localisation and the formation of pleiotropic spindle abnormalities and misaligned chromosomes [30]. BRAF isoforms have also been detected in nuclear fractions of the rat forebrain and cerebellum [31] with a recent investigation identifying BRAF in the nucleus of skeletal muscle cells after activation, where it was found to interact with and phosphorylate PAX3 leading to enhancement of MET activity, a requirement for limb muscle precursor cell migration [32]. However, the relevance of these alternative locations for BRAF and their role in downstream MEK/ERK signaling and BRAF-driven oncogenesis has not been fully explored as yet.…”

Section: Introductionmentioning

confidence: 99%

“…Despite the overwhelming evidence supporting a cytoplasmic location of RAF proteins and their translocation to the plasma membrane upon activation [16, 26], there are reports of alternative locations within the cell. BRAF in particular has been detected in mitochondria [27], Golgi [28, 29], the mitotic spindle [30] and the nucleus [31, 32], and this compartmentalisation is associated with distinct biological outcomes in some circumstances [27, 30, 32]. For example, a portion of BRAF has been detected at spindle poles and kinetochores in mitotic HeLa cells and knockdown of BRAF using siRNA resulted in early exit of cells from mitosis, perturbation of Mps1 localisation and the formation of pleiotropic spindle abnormalities and misaligned chromosomes [30].…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Over-expressed, N-terminally truncated BRAF is detected in the nucleus of cells with nuclear phosphorylated MEK and ERK

Hey

Andreadi

Noble

et al. 2018

Heliyon

BRAF is a cytoplasmic protein kinase, which activates the MEK-ERK signalling pathway. Deregulation of the pathway is associated with the presence of BRAF mutations in human cancer, the most common being V600EBRAF, although structural rearrangements, which remove N-terminal regulatory sequences, have also been reported. RAF-MEK-ERK signalling is normally thought to occur in the cytoplasm of the cell. However, in an investigation of BRAF localisation using fluorescence microscopy combined with subcellular fractionation of Green Fluorescent Protein (GFP)-tagged proteins expressed in NIH3T3 cells, surprisingly, we detected N-terminally truncated BRAF (ΔBRAF) in both nuclear and cytoplasmic compartments. In contrast, ΔCRAF and full-length, wild-type BRAF (WTBRAF) were detected at lower levels in the nucleus while full-length V600EBRAF was virtually excluded from this compartment. Similar results were obtained using ΔBRAF tagged with the hormone-binding domain of the oestrogen receptor (hbER) and with the KIAA1549-ΔBRAF translocation mutant found in human pilocytic astrocytomas. Here we show that GFP-ΔBRAF nuclear translocation does not involve a canonical Nuclear Localisation Signal (NLS), but is suppressed by N-terminal sequences. Nuclear GFP-ΔBRAF retains MEK/ERK activating potential and is associated with the accumulation of phosphorylated MEK and ERK in the nucleus. In contrast, full-length GFP-WTBRAF and GFP-V600EBRAF are associated with the accumulation of phosphorylated ERK but not phosphorylated MEK in the nucleus. These data have implications for cancers bearing single nucleotide variants or N-terminal deleted structural variants of BRAF.

“…Lbx1 plays a similar role in zebrafish where FGF signalling from within the fin bud controls the Lbx1 phosphorylation state, which is important for MMP function in the zebrafish pectoral fin development [19]. The RAS-RAF pathway, particularly BRAF, positively regulates and directly activates Pax3 and subsequently C-met to drive MMP derived limb muscle formation [20]. The MMPs are also guided through the lateral mesoderm toward the limb bud by the chemokine receptor CXCR4 which enable the MMPs to be attracted to the ligand XCL12/SDF-1 expressing limb mesenchyme [21–23].…”

Section: Introductionmentioning

confidence: 99%

The zebrafish HGF receptor met controls migration of myogenic progenitor cells in appendicular development

et al. 2019

The hepatocyte growth factor receptor C-met plays an important role in cellular migration, which is crucial for many developmental processes as well as for cancer cell metastasis. C-met has been linked to the development of mammalian appendicular muscle, which are derived from migrating muscle progenitor cells (MMPs) from within the somite. Mammalian limbs are homologous to the teleost pectoral and pelvic fins. In this study we used Crispr/Cas9 to mutate the zebrafish met gene and found that the MMP derived musculature of the paired appendages was severely affected. The mutation resulted in a reduced muscle fibre number, in particular in the pectoral abductor, and in a disturbed pectoral fin function. Other MMP derived muscles, such as the sternohyoid muscle and posterior hypaxial muscle were also affected in met mutants. This indicates that the role of met in MMP function and appendicular myogenesis is conserved within vertebrates.