Project description:Analyze effect of Raf1 S259A on gene expression in HUVEC Total RNA obtained from HUVEC infected with empty lentiviral vector pLVX-IRES-puro (control) or that expressing human RAF1 WT (WT) or RAF1 S259A (S259A).
Project description:Arterial morphogenesis is one of the most critical events during embryonic vascular development. Although arterial fate specification is mainly controlled by the Notch signaling pathway, arterial-venous patterning is modulated by a number of guidance factors. How these pathways are regulated is still largely unknown. Here, we demonstrate that endothelial activation of RAF1/extracellular signal-regulated kinase (ERK) pathway regulates arterial morphogenesis and arterial-venous patterning via ?/Notch and semaphorin signaling. Introduction of a single amino acid RAF1 mutant (RAF1 Ser259Ala), which renders it resistant to inhibition by phosphorylation, into endothelial cells in vitro induced expression of virtually the entire embryonic arteriogenic program and activated semaphorin 6A-dependent endothelial cell-cell repulsion. In vivo, endothelial-specific expression of RAF1(S259A) during development induced extensive arterial morphogenesis both in the yolk sac and the embryo proper and disrupted arterial-venous patterning. Our results suggest that endothelial ERK signaling is critical for both arteriogenesis and arterial-venous patterning and that RAF1 Ser(259) phosphorylation plays a critical role in preventing unopposed ERK activation.
Project description:In this study we performed gene transcription analysis of parental MCF7 cells and MCF7 engineered to overexpress oncoprotein RAF1. We demonstrate that overexpression of RAF1 leads to development of ivasive phenotype.
Project description:HUVEC-FUCCI cells were used to demonstrate that different endothelial cell cycle states provide distict windows of opportunity for gene expression in response to extrinsic signals. HUVEC-FUCCI were FACS-isolated into three different cell cycle states. Peptide digests from the resulting lysates showed differentially expressed proteins among the three cell cycles. These studies show that endothelial cell cycle state determines the propensity for arterial vs. venous fate specification.
Project description:Noonan syndrome (NS) is caused by mutations in RAS/ERK pathway genes, and is characterized by craniofacial, growth, cognitive and cardiac defects. NS patients with kinase-activating RAF1 alleles typically develop pathological left ventricular hypertrophy (LVH), which is reproduced in Raf1 L613V/+ knock-in mice. Here, using inducible Raf1 L613V expression, we show that LVH results from the interplay of cardiac cell types. Cardiomyocyte Raf1 L613V enhances Ca 2+ sensitivity and cardiac contractility without causing hypertrophy. Raf1 L613V expression in cardiomyocytes or activated fibroblasts exacerbates pressure overload-evoked fibrosis. Endothelial/endocardial (EC) Raf1 L613V causes cardiac hypertrophy without affecting contractility. Co-culture and neutralizing antibody experiments reveal a cytokine hierarchy (TNFα->IL6) from Raf1 L613V -expressing ECs that drives cardiomyocyte hypertrophy in vitro. Furthermore, post-natal TNFα inhibition normalizes the increased wall thickness and cardiomyocyte hypertrophy in vivo. We conclude that NS cardiomyopathy involves cardiomyocytes, ECs, and fibroblasts, TNFα/IL6 signaling components represent potential therapeutic targets, and abnormal EC signaling might contribute to other forms of LVH.
Project description:RAF1 is a crucial element of the MAPK/ERK signaling pathway. TurboID, an efficient promiscuous biotin ligase, enables the biotinylation of proteins in the vicinity, thus facilitating the study of complex cellular processes. In this study, we utilized TurboID fused to RAF1WT, RAF1K375M (kinase inactive) and RAF1CAT(catalytic domain, constitutively active) to investigate the intricate interaction network of RAF1 under various conditions.
Project description:RAF kinases play major roles in cancer. BRAFV600E mutants drive ~6% of human cancers. Potent kinase inhibitors exist but show variable effects in different cancer types, sometimes even inducing paradoxical RAF kinase activation. Both paradoxical activation and drug resistance are frequently due to enhanced dimerization between RAF1 and BRAF, which maintains or restores the activity of the downstream MEK-ERK pathway. Here, using quantitative proteomics we mapped the interactomes of RAF1 monomers, RAF1-BRAF and RAF1-BRAFV600E dimers identifying and quantifying >1,000 proteins. In addition, we examined the effects of vemurafenib and sorafenib, two different types of clinically used RAF inhibitors. Using regression analysis to compare different conditions we found a large overlapping core interactome but also distinct condition specific differences. Given that RAF proteins have kinase independent functions such dynamic interactome changes could contribute to their functional diversification. Analysing this dataset may provide a deeper understanding of RAF signalling and mechanisms of resistance to RAF inhibitors.
Project description:Sprouting angiogenesis is a multistep process that involves endothelial cell activation, basement membrane degradation, proliferation, lumen formation, and stabilization. In this study, we identified annexin 2 as a regulator of endothelial morphogenesis using a three-dimensional in vitro model where sprouting angiogenesis was driven by sphingosine 1-phosphate and angiogenic growth factors. We observed that sphingosine 1-phosphate triggered annexin 2 translocation from the cytosol to the plasma membrane and its association with vascular endothelial (VE)-cadherin. In addition, annexin 2 depletion attenuated Akt activation, which was associated with increased phosphorylation of VE-cadherin and endothelial barrier leakage. Disrupting homotypic VE-cadherin interactions with EGTA, antibodies to the extracellular domain of VE-cadherin, or gene silencing all resulted in decreased Akt (but not Erk1/2) activation. Furthermore, expression of constitutively active Akt restored reduced endothelial sprouting responses observed with annexin 2 and VE-cadherin knockdown. Collectively, we report that annexin 2 regulates endothelial morphogenesis through an adherens junction-mediated pathway upstream of Akt.
Project description:VEGF is the key growth factor regulating arterial morphogenesis. However, molecular events involved in this process have not been elucidated. Synectin null mice demonstrate impaired VEGF signaling and a marked reduction in arterial morphogenesis. Here, we show that this occurs due to delayed trafficking of VEGFR2-containing endosomes that exposes internalized VEGFR2 to selective dephosphorylation by PTP1b on Y(1175) site. Synectin involvement in VEGFR2 intracellular trafficking requires myosin-VI, and myosin-VI knockout in mice or knockdown in zebrafish phenocopy the synectin null phenotype. Silencing of PTP1b restores VEGFR2 activation and significantly recovers arterial morphogenesis in myosin-VI(-/-) knockdown zebrafish and synectin(-/-) mice. We conclude that activation of the VEGF-mediated arterial morphogenesis cascade requires phosphorylation of the VEGFR2 Y(1175) site that is dependent on trafficking of internalized VEGFR2 away from the plasma membrane via a synectin-myosin-VI complex. This key event in VEGF signaling occurs at an intracellular site and is regulated by a novel endosomal trafficking-dependent process.