scholarly journals A Fyn - specific biosensor reveals localized, pulsatile kinase activity and spatially regulated signaling crosstalk

2019 ◽  
Author(s):  
Ananya Mukherjee ◽  
Randhir Singh ◽  
Sreeram Udayan ◽  
Sayan Biswas ◽  
Purushotham Pothula ◽  
...  
Keyword(s):  
Growth Factor ◽  
Cell Fate ◽  
Kinase Activity ◽  
Activity Patterns ◽  
Live Cells ◽  
Structural Basis ◽  
Cell Physiology ◽  
Growth Factor Signaling ◽  
Protein Activity ◽  
Signaling Crosstalk

AbstractCell behavior is controlled through spatio-temporally localized protein activity. Despite unique and often contradictory roles played by Src-family-kinases (SFKs) in regulating cell physiology, activity patterns of individual SFKs have remained elusive. Here, we report a biosensor for specifically visualizing active conformation of SFK-Fyn in live cells. We deployed combinatorial library screening to isolate a binding-protein (F29) targeting activated Fyn. Nuclear-magnetic-resonance (NMR) analysis provides the structural basis of F29 specificity for Fyn over homologous SFKs. Using F29, we engineered a sensitive, minimally-perturbing fluorescence-resonance-energy-transfer (FRET) biosensor (FynSensor) that reveals cellular Fyn activity to be spatially localized, pulsatile and sensitive to adhesion/integrin signaling. Strikingly, growth factor stimulation further enhanced Fyn activity in pre-activated intracellular zones. However, inhibition of focal-adhesion-kinase activity not only attenuates Fyn activity, but abolishes growth-factor modulation. FynSensor imaging uncovers spatially-organized, sensitized signaling clusters, direct crosstalk between integrin and growth-factor-signaling, and clarifies how compartmentalized Src-kinase activity may drive cell fate.

scholarly journals A Fyn biosensor reveals pulsatile, spatially localized kinase activity and signaling crosstalk in live mammalian cells

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Ananya Mukherjee ◽  
Randhir Singh ◽  
Sreeram Udayan ◽  
Sayan Biswas ◽  
Pothula Purushotham Reddy ◽  
...  
Keyword(s):  
Growth Factor ◽  
Cell Fate ◽  
Mammalian Cells ◽  
Kinase Activity ◽  
Live Cells ◽  
Structural Basis ◽  
Cell Physiology ◽  
Growth Factor Signaling ◽  
Protein Activity ◽  
Signaling Crosstalk

Cell behavior is controlled through spatio-temporally localized protein activity. Despite unique and often contradictory roles played by Src-family-kinases (SFKs) in regulating cell physiology, activity patterns of individual SFKs have remained elusive. Here, we report a biosensor for specifically visualizing active conformation of SFK-Fyn in live cells. We deployed combinatorial library screening to isolate a binding-protein (F29) targeting activated Fyn. Nuclear-magnetic-resonance (NMR) analysis provides the structural basis of F29 specificity for Fyn over homologous SFKs. Using F29, we engineered a sensitive, minimally-perturbing fluorescence-resonance-energy-transfer (FRET) biosensor (FynSensor) that reveals cellular Fyn activity to be spatially localized, pulsatile and sensitive to adhesion/integrin signaling. Strikingly, growth factor stimulation further enhanced Fyn activity in pre-activated intracellular zones. However, inhibition of focal-adhesion-kinase activity not only attenuates Fyn activity, but abolishes growth-factor modulation. FynSensor imaging uncovers spatially organized, sensitized signaling clusters, direct crosstalk between integrin and growth-factor-signaling, and clarifies how compartmentalized Src-kinase activity may drive cell fate.

Abstract 168: Contribution of Vascular Endothelial Growth Factor Signaling to Fate Specification in Cardiomyocytes

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Taylor Y Lu ◽  
Courtney K Domigan ◽  
Vaspour Antanesian ◽  
Yasuhiro Nakashima ◽  
Atsushi Nakano ◽  
...  
Keyword(s):  
Vascular Endothelial Growth Factor ◽  
Growth Factor ◽  
Cell Fate ◽  
Heart Development ◽  
Embryonic Stem ◽  
Growth Factor Signaling ◽  
Vascular Endothelial ◽  
Rt Pcr ◽  
Cardiac Morphogenesis ◽  
Endothelial Growth Factor

Vascular endothelial growth factor (VEGF) is one of the pivotal proangiogenic growth factors that has long contributed to our knowledge of blood vessel and circulatory maintenance as well as angiogenesis in both pathology and pathophysiology. However, the non-canonical functions of VEGF in cardiac morphogenesis have not been well characterized. Here, we examined how VEGF regulates cardiomyocyte cell fate. Using chimeric embryos harboring both wild type and VEGF-null embryonic stem cells, we observed that derivatives of VEGF null cells were preferentially recruited to the atrium of the heart in comparison to the ventricles. To further provide physiologic context of this finding, we used reporter-LacZ staining and RT-PCR and found that endogenous VEGF was indeed expressed at much lower levels in the atrium but highly expressed in the ventricle early in cardiac morphogenesis. These data lead to our hypothesis that cell-autonomous expression of VEGF is a determinant of atrial vs. ventricular cardiomyocyte cell fate. To test this hypothesis, we used a VEGF knock-in mouse model of Sm22Cre x Rosa 26 VEGF. VEGF overexpression in cardiomyocytes (and smooth muscle) at E8.5 resulted in lethality by P1 and thickened atrial and ventricular walls in mutant embryos as characterized by histology (H&E, IF). We further explored the molecular changes underlying this phenotype via microarray and RT-PCR and find disruptions in molecular markers necessary for wall development, specifically: Notch-1, BMP10, Nrg-1. Taken together, our data indicates that aberrant embryonic VEGF signaling disrupts several critical signaling pathways and that overexpression leads to disruption of cardiomyocyte proliferation and cardiac morphogenesis. These findings add to the foundation of better understanding heart development, laying the groundwork for future therapy of congenital and acquired cardiac disease.

Resistance to endocrine therapy in breast cancer: exploiting estrogen receptor/growth factor signaling crosstalk

2006 ◽  
Vol 13 (Supplement_1) ◽  
pp. S15-S24 ◽  
Author(s):  
Suleiman Massarweh ◽  
Rachel Schiff
Keyword(s):  
Breast Cancer ◽  
Estrogen Receptor ◽  
Growth Factor ◽  
Endocrine Therapy ◽  
Acquired Resistance ◽  
Growth Factor Signaling ◽  
Breast Cancer Patients ◽  
Signaling Crosstalk ◽  
Receptor Modulator ◽  
Signal Transduction Inhibitors

Targeting the estrogen receptor (ER) is the oldest form of molecular targeted therapy, and the widespread use of the selective estrogen receptor modulator tamoxifen in breast cancer is responsible for major improvements in cure rates, quality of life, and disease prevention in the last 25 years. Newer forms of endocrine therapy now available for the management of endocrine responsive breast cancer include a new generation of aromatase inhibitors, which lower the estrogen ligand for ER, and pure ER antagonists which destroy the receptor. Despite these recent clinical advances, intrinsic and acquired resistance to these endocrine therapies is still a common feature that limits the success of this therapeutic strategy. Recent research into the molecular biology of ER signaling has revealed a remarkably complex interactive signaling with other growth factor signaling pathways in breast cancer cells, potentially explaining some of the reasons behind endocrine therapy action as well as resistance. This view of a more complex ER signaling system has uncovered new molecular targets which, if present in a cancer cell, might be additionally targeted using various signal transduction inhibitors to overcome or prevent resistance to endocrine therapy. In addition, the dynamic inverse relationship between the expression of ER and growth factor receptors brings more excitement to the potential of restoring ER expression in apparently ER-negative cells by inhibition of growth factor signaling. Ongoing clinical trials of endocrine therapy combined with growth factor pathway inhibitors or their downstream signaling elements promise to further improve the present care for breast cancer patients.

scholarly journals FGF signaling dynamics regulates epithelial patterning and morphogenesis

2020 ◽  
Author(s):  
Jakub Sumbal ◽  
Tereza Vranova ◽  
Zuzana Koledova
Keyword(s):  
Growth Factor ◽  
Cell Fate ◽  
Mammary Epithelium ◽  
Time Lapse ◽  
Branching Morphogenesis ◽  
Tissue Level ◽  
Epithelial Tissue ◽  
Growth Factor Signaling ◽  
Fgf Signaling ◽  
Signaling Dynamics

SummarySingle cell assays revealed that growth factor signaling dynamics is actively sensed by a cell and ultimately controls cell fate. However, the effects of growth factor signaling dynamics at the tissue level have been unknown. We used mammary epithelial organoids, time-lapse imaging, fibroblast growth factor 2 (FGF2) variants of different stabilities, mathematical modeling, and perturbation analysis to study the role of FGF2 signaling dynamics in epithelial morphogenesis. We found that fluctuant and sustained FGF signaling dynamics induced distinct morphological and functional states of mammary epithelium through differential employment of intracellular effectors ERK and AKT. ERK activity domains determined epithelial branch size, while AKT activity drove epithelial stratification. Furthermore, FGF signaling dynamics affected epithelial tissue mechanoresponsiveness to extracellular matrix, thereby impinging upon branch elongation. Our study provides new insights into regulation of epithelial patterning and branching morphogenesis by FGF signaling dynamics and into downstream signaling effectors that regulate cellular outcomes.

scholarly journals It Takes Two to Tango: Endothelial TGFβ/BMP Signaling Crosstalk with Mechanobiology

Cells ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 1965 ◽  
Author(s):  
Christian Hiepen ◽  
Paul-Lennard Mendez ◽  
Petra Knaus
Keyword(s):  
Shear Stress ◽  
Growth Factor ◽  
Transforming Growth Factor Beta ◽  
Molecular Mechanisms ◽  
Transforming Growth Factor ◽  
Research Field ◽  
Bmp Signaling ◽  
Growth Factor Signaling ◽  
Mechanical Stimuli ◽  
Signaling Crosstalk

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta (TGFβ) superfamily of cytokines. While some ligand members are potent inducers of angiogenesis, others promote vascular homeostasis. However, the precise understanding of the molecular mechanisms underlying these functions is still a growing research field. In bone, the tissue in which BMPs were first discovered, crosstalk of TGFβ/BMP signaling with mechanobiology is well understood. Likewise, the endothelium represents a tissue that is constantly exposed to multiple mechanical triggers, such as wall shear stress, elicited by blood flow or strain, and tension from the surrounding cells and to the extracellular matrix. To integrate mechanical stimuli, the cytoskeleton plays a pivotal role in the transduction of these forces in endothelial cells. Importantly, mechanical forces integrate on several levels of the TGFβ/BMP pathway, such as receptors and SMADs, but also global cell-architecture and nuclear chromatin re-organization. Here, we summarize the current literature on crosstalk mechanisms between biochemical cues elicited by TGFβ/BMP growth factors and mechanical cues, as shear stress or matrix stiffness that collectively orchestrate endothelial function. We focus on the different subcellular compartments in which the forces are sensed and integrated into the TGFβ/BMP growth factor signaling.

scholarly journals Growth factor-mediated coupling between lineage size and cell fate choice underlies robustness of mammalian development

2019 ◽  
Author(s):  
Néstor Saiz ◽  
Laura Mora-Bitria ◽  
Shahadat Rahman ◽  
Hannah George ◽  
Jeremy P Herder ◽  
...  
Keyword(s):  
Growth Factor ◽  
Cell Fate ◽  
Population Level ◽  
Local Concentration ◽  
Additional Parameter ◽  
Growth Factor Signaling ◽  
Cell Type ◽  
Mammalian Embryo ◽  
Cell Fate Decisions

SummaryPrecise control and maintenance of the size of cell populations is fundamental for organismal development and homeostasis. The three cell types that comprise the mammalian blastocyst-stage embryo are generated in precise proportions and over a short time, suggesting a size control mechanism ensures a reproducible outcome. Guided by experimental observations, we developed a minimal mathematical model that shows growth factor signaling is sufficient to guarantee this robustness. The model anticipates, without additional parameter fitting, the response of the embryo to perturbations in its lineage composition. We experimentally added lineage-restricted cells to the epiblast both in vivo and in silico, which resulted in a shift of the fate of progenitors away from the supernumerary cell type, while eliminating cells using laser ablation biased the specification of progenitors towards the targeted cell type. Finally, we show that FGF4 couples cell fate decisions to lineage composition through changes in local concentration of the growth factor. Our results provide a basis for the regulative abilities of the mammalian embryo and reveal how, in a self-organizing system, individual cell fate decisions are coordinated at the population level to robustly generate tissues in the right proportions.

scholarly journals Structural Basis for Activation of Fibroblast Growth Factor Signaling by Sucrose Octasulfate

2002 ◽  
Vol 22 (20) ◽  
pp. 7184-7192 ◽  
Author(s):  
Brian K. Yeh ◽  
Anna V. Eliseenkova ◽  
Alexander N. Plotnikov ◽  
David Green ◽  
Jared Pinnell ◽  
...  
Keyword(s):  
Growth Factor ◽  
Fibroblast Growth Factor ◽  
Protein Interactions ◽  
Therapeutic Potential ◽  
Structural Basis ◽  
Growth Factor Signaling ◽  
Fibroblast Growth ◽  
Fgf Signaling ◽  
Receptor Dimerization ◽  
Sucrose Octasulfate

ABSTRACT Sucrose octasulfate (SOS) is believed to stimulate fibroblast growth factor (FGF) signaling by binding and stabilizing FGFs. In this report, we show that SOS induces FGF-dependent dimerization of FGF receptors (FGFRs). The crystal structure of the dimeric FGF2-FGFR1-SOS complex at 2.6-Å resolution reveals a symmetric assemblage of two 1:1:1 FGF2-FGFR1-SOS ternary complexes. Within each ternary complex SOS binds to FGF and FGFR and thereby increases FGF-FGFR affinity. SOS also interacts with the adjoining FGFR and thereby promotes protein-protein interactions that stabilize dimerization. This structural finding is supported by the inability of selectively desulfated SOS molecules to promote receptor dimerization. Thus, we propose that SOS potentiates FGF signaling by imitating the dual role of heparin in increasing FGF-FGFR affinity and promoting receptor dimerization. Hence, the dimeric FGF-FGFR-SOS structure substantiates the recently proposed “two-end” model, by which heparin induces FGF-FGFR dimerization. Moreover, the FGF-FGFR-SOS structure provides an attractive template for the development of easily synthesized SOS-related heparin agonists and antagonists that may hold therapeutic potential.

scholarly journals Regulation of Phosphoinositide 3-Kinase by Its Intrinsic Serine Kinase Activity In Vivo

2004 ◽  
Vol 24 (3) ◽  
pp. 966-975 ◽  
Author(s):  
Lazaros C. Foukas ◽  
Caroline A. Beeton ◽  
Jorgen Jensen ◽  
Wayne A. Phillips ◽  
Peter R. Shepherd
Keyword(s):  
Growth Factor ◽  
Kinase Activity ◽  
Catalytic Subunit ◽  
Protein Kinase Activity ◽  
Growth Factor Signaling ◽  
Serine Kinase ◽  
Lipid Kinase ◽  
Phosphoinositide 3 Kinase

ABSTRACT One potentially important mechanism for regulating class Ia phosphoinositide 3-kinase (PI 3-kinase) activity is autophosphorylation of the p85α adapter subunit on Ser608 by the intrinsic protein kinase activity of the p110 catalytic subunit, as this downregulates the lipid kinase activity in vitro. Here we investigate whether this phosphorylation can occur in vivo. We find that p110α phosphorylates p85α Ser608 in vivo with significant stoichiometry. However, p110β is far less efficient at phosphorylating p85α Ser608, identifying a potential difference in the mechanisms by which these two isoforms are regulated. The p85α Ser608 phosphorylation was increased by treatment with insulin, platelet-derived growth factor, and the phosphatase inhibitor okadaic acid. The functional effects of this phosphorylation are highlighted by mutation of Ser608, which results in reduced lipid kinase activity and reduced association of the p110α catalytic subunit with p85α. The importance of this phosphorylation was further highlighted by the finding that autophosphorylation on Ser608 was impaired, while lipid kinase activity was increased, in a p85α mutant recently discovered in human tumors. These results provide the first evidence that phosphorylation of Ser608 plays a role as a shutoff switch in growth factor signaling and contributes to the differences in functional properties of different PI 3-kinase isoforms in vivo.

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