A Direct Role for ATP1A1 in Unconventional Secretion of Fibroblast Growth Factor 2

Zacherl, Sonja; Venuta, Giuseppe La; Müller, Hans‐Michael; Wegehingel, Sabine; Dimou, Eleni; Sehr, Peter; Lewis, Joe; Erfle, Holger; Pepperkok, Rainer; Nickel, Walter

doi:10.1074/jbc.m114.590067

Cited by 54 publications

(72 citation statements)

References 40 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This process involves (i) membrane recruitment at the inner leaflet mediated by the phosphoinositide PI(4,5)P 2 (1, 2, 21, 22), (ii) FGF2 oligomerization and membrane pore formation (9,11,23,24,30), and (iii) extracellular trapping mediated by membraneproximal heparan sulfate proteoglycans (25,26). In addition, Tec kinase (9, 31) and ATP1A1 (12,(32)(33)(34), two additional factors physically associated with the plasma membrane, have been shown to play critical roles in the unconventional secretory pathway of FGF2 (12). Whereas ATP1A1 is an integral membrane protein, Tec kinase contains a PH domain and associates with the inner leaflet via recruitment by the phosphoinositide PI(3,4,5)P 3 (36,37).…”

Section: Discussionmentioning

confidence: 99%

“…FGF2 was expressed in Escherichia coli, purified to homogeneity (23,24), and covalently coupled to epoxy beads (34). Various constructs of Tec kinase were expressed and purified from insect cells (Fig.…”

Section: Biochemical Characterization Of Fgf2 Binding To Tecmentioning

confidence: 99%

“…Recently, these hallmarks of FGF2 secretion have also been implicated in unconventional secretion of HIV-Tat (HIV trans-activator of transcription) (12,(27)(28)(29)(30). Based on a genome-wide RNAi screening approach, two additional factors physically associated with the plasma membrane have been identified to play a role in FGF2 secretion, Tec kinase (9,11,12,24,31) and ATP1A1 (12,(32)(33)(34), the ␣ subunit of the Na/K-ATPase (35). Although the precise role of ATP1A1 in FGF2 membrane translocation into the extracellular space is unknown, Tec kinase was shown to directly interact with FGF2, resulting in phosphorylation of tyrosine 81 (9,31).…”

mentioning

confidence: 99%

“…9. Cell proliferation was quantified in real time, employing an IncuCyte Zoom live cell imaging microscope (Essen BioScience), providing an absolute quantification of fluorescent cell nuclei with kinetic resolution (34). In this way, cell proliferation was monitored for 72 h (with measurements conducted every 2 h) under the conditions indicated (Fig.…”

mentioning

confidence: 99%

“…Following the determination of affinity between FGF2 and Tec kinase, an Alpha protein-protein interaction assay (34) was used to screen a library of 79,000 compounds to identify small molecules inhibitors that block binding of FGF2 to Tec kinase. Here we report the structure of three active, structurally related compounds that (i) block the interaction of FGF2 with Tec kinase, (ii) prevent phosphorylation of FGF2 by Tec kinase in vitro and in a cellular context, and (iii) inhibit unconventional secretion of FGF2 from cells.…”

mentioning

confidence: 99%

See 4 more Smart Citations

Small Molecule Inhibitors Targeting Tec Kinase Block Unconventional Secretion of Fibroblast Growth Factor 2

Venuta

Wegehingel

Sehr

et al. 2016

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Fibroblast growth factor 2 (FGF2) is a potent mitogen promoting both tumor cell survival and tumor-induced angiogenesis. It is secreted by an unconventional secretory mechanism that is based upon direct translocation across the plasma membrane. Key steps of this process are (i) phosphoinositide-dependent membrane recruitment, (ii) FGF2 oligomerization and membrane pore formation, and (iii) extracellular trapping mediated by membrane-proximal heparan sulfate proteoglycans. Efficient secretion of FGF2 is supported by Tec kinase that stimulates membrane pore formation based upon tyrosine phosphorylation of FGF2. Here, we report the biochemical characterization of the direct interaction between FGF2 and Tec kinase as well as the identification of small molecules that inhibit (i) the interaction of FGF2 with Tec, (ii) tyrosine phosphorylation of FGF2 mediated by Tec in vitro and in a cellular context, and (iii) unconventional secretion of FGF2 from cells. We further demonstrate the specificity of these inhibitors for FGF2 because tyrosine phosphorylation of a different substrate of Tec is unaffected in their presence. Building on previous evidence using RNA interference, the identified compounds corroborate the role of Tec kinase in unconventional secretion of FGF2. In addition, they are valuable lead compounds with great potential for drug development aiming at the inhibition of FGF2-dependent tumor growth and metastasis.Although the majority of secretory proteins carry signal peptides for endoplasmic reticulum/Golgi-dependent secretion, a set of important growth factors and cytokines involved in tumor-induced angiogenesis and inflammatory responses makes use of alternative routes. These processes have collectively been termed "unconventional protein secretion" (1-5). FGF2 and IL1␤ are prominent examples for secretory proteins that make use of such pathways (4 -12). The molecular mechanism by which IL1␤ is secreted from cells is debated and may differ in some aspects between various kinds of immune cells (4,5,10,(13)(14)(15)(16). By contrast, the mechanism of the unconventional secretory pathway of FGF2 is emerging with great molecular detail (12). It is based upon direct translocation of folded species of FGF2 across plasma membranes (9,(17)(18)(19)(20). Hallmarks of this process are (i) FGF2 recruitment at the inner leaflet of the plasma membrane mediated by the phosphoinositide PI(4,5)P 2 2 (9, 21, 22), (ii) FGF2 oligomerization and membrane pore formation (11,12,23,24), and (iii) extracellular trapping of FGF2 mediated by membrane-proximal heparan sulfate proteoglycans (1,2,25,26). Recently, these hallmarks of FGF2 secretion have also been implicated in unconventional secretion of HIV-Tat (HIV trans-activator of transcription) (12, 27-30). Based on a genome-wide RNAi screening approach, two additional factors physically associated with the plasma membrane have been identified to play a role in FGF2 secretion, Tec kinase (9, 11, 12, 24, 31) and ATP1A1 (12, 32-34), the ␣ subunit of the Na/K-ATPase (35). Although t...

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Biochemical Characterization Of Fgf2 Binding To Tecmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

Small Molecule Inhibitors Targeting Tec Kinase Block Unconventional Secretion of Fibroblast Growth Factor 2

Venuta

Wegehingel

Sehr

et al. 2016

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

A time‐resolved live cell imaging assay to identify small molecule inhibitors of FGF2 signaling

et al. 2019

Self Cite

View full text Add to dashboard Cite

Fibroblast growth factor 2 (FGF2) is a cell survival factor with crucial functions in tumor‐induced angiogenesis. Here, we describe a novel time‐resolved FGF2 signaling assay based upon live cell imaging of neuroblastoma cells. To validate this system, we tested 8960 small molecules for inhibition of FGF2 signaling with kinetic resolution. Hit compounds were validated in dose‐response experiments for FGF2 signaling, FGF receptor antagonism, downstream ERK phosphorylation and FGF2‐dependent chemoresistance in a cellular leukemia model system. The new screening system for FGF2 signaling inhibitors has unique features, deselecting compounds with pleiotropic effects on cell proliferation and, along with the experimental pipeline reported, great potential for the discovery of new classes of FGF2 signaling inhibitors that block FGF2 dependent tumor cell survival.

show abstract

Tyrosine Kinase Expressed in Hepatocellular Carcinoma, TEC, Controls Pluripotency and Early Cell Fate Decisions of Human Pluripotent Stem Cells via Regulation of Fibroblast Growth Factor-2 Secretion

et al. 2017

Self Cite

View full text Add to dashboard Cite

Human pluripotent stem cells (hPSC) require signaling provided by fibroblast growth factor (FGF) receptors. This can be initiated by the recombinant FGF2 ligand supplied exogenously, but hPSC further support their niche by secretion of endogenous FGF2. In this study, we describe a role of tyrosine kinase expressed in hepatocellular carcinoma (TEC) kinase in this process. We show that TEC-mediated FGF2 secretion is essential for hPSC self-renewal, and its lack mediates specific differentiation. Following both short hairpin RNA- and small interfering RNA-mediated TEC knockdown, hPSC secretes less FGF2. This impairs hPSC proliferation that can be rescued by increasing amounts of recombinant FGF2. TEC downregulation further leads to a lower expression of the pluripotency markers, an improved priming towards neuroectodermal lineage, and a failure to develop cardiac mesoderm. Our data thus demonstrate that TEC is yet another regulator of FGF2-mediated hPSC pluripotency and differentiation. Stem Cells 2017;35:2050-2059.

show abstract

A Direct Role for ATP1A1 in Unconventional Secretion of Fibroblast Growth Factor 2

Cited by 54 publications

References 40 publications

Small Molecule Inhibitors Targeting Tec Kinase Block Unconventional Secretion of Fibroblast Growth Factor 2

Small Molecule Inhibitors Targeting Tec Kinase Block Unconventional Secretion of Fibroblast Growth Factor 2

A time‐resolved live cell imaging assay to identify small molecule inhibitors of FGF2 signaling

Tyrosine Kinase Expressed in Hepatocellular Carcinoma, TEC, Controls Pluripotency and Early Cell Fate Decisions of Human Pluripotent Stem Cells via Regulation of Fibroblast Growth Factor-2 Secretion

Contact Info

Product

Resources

About