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

Abstract

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...

The α1 subunit of the Na,K-ATPase acts upstream of PI(4,5)P2 facilitating unconventional secretion of Fibroblast Growth Factor 2 from tumor cells

“…Using an untagged version of FGF2 as a competitor for the interaction of His-tagged FGF2 and GST-tagged α1 constructs, the AlphaScreen ® assay also allowed for a quantitative comparison of the affinities between FGF2 and α1-CD1-3, α1-CD3 as well as α1-subCD3, respectively [ Fig. 2C; 15,41 ]. As reported previously, FGF2 binds to the complete cytoplasmic domain of α1 with an apparent KD of about 0.8 µM 15 .…”

“…11C and 12D). Following induction of protein expression with doxycycline, a well-established cell surface biotinylation assay was used to quantify the extracellular amounts of the FGF2-GFP fusion proteins indicated 20,23,41,43 . These experiments revealed that a K54/60E substitution alone caused a significant impairment of FGF2-GFP transport to the cell surface with only 60% efficiency compared to the wild-type form of FGF2-GFP ( Fig.…”

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

“…Western blot was carried out as described before . The following antibodies were used: TEC, anti‐pY‐FGF2 , Actin (#3700), Nanog (#3580, Cell Signaling Technology, Danvers, MA), Oct4 (sc‐8629, Santa Cruz Biotechnology, Santa Cruz, CA, https://www.scbt.com/), FGF2 (F6162), SOX17 (AF1924, R&D Systems), FOX2A (H00003170‐M12, Novus Biologicals Europe, Abingdon, UK, https://www.novusbio.com/), α‐tubulin (11–250‐C100, Exbio, Prag, CZE, http://www.exbio.cz/), SOX2 (ab79351, Abcam, Cambridge, UK, http://www.abcam.com/), and PAX6 (DSHB Hybridoma Product PAX6, DSHB, Iowa City, IA, http://dshb.biology.uiowa.edu/). The protein bands were quantified using ImageJ (http://imagej.nih.gov/ij/).…”

The molecular mechanism underlying unconventional secretion of Fibroblast Growth Factor 2 from tumour cells

“…Tec kinase itself is recruited to the inner leaflet via its N‐terminal pleckstrin homology domain in a phosphatidylinositol‐3,4,5‐triphosphate [PI(3,4,5)P 3 ]‐dependent manner (Shirai et al., ). The interaction of Tec kinase with FGF2 results in phosphorylation of FGF2 at tyrosine 81 (Figure ; highlighted in red) (Ebert et al., ; La Venuta et al., ). This modification is believed to have regulatory function tuning the efficiency of FGF2 secretion from cells (La Venuta et al., ).…”