Mechanisms Underlying Triiodothyronine Formation Within Thyroglobulin in Hyperstimulated Thyrocytes

CAROLINA MARÍA IBÁÑEZ PADILLA; HÉCTOR TARGOVNIK; PETER ARVAN; EZEQUIEL SANTIAGO ARÉVALOS; CINTIA E. CITTERIO

Mar del Plata

Congreso; LXIII Reunión de la Sociedad Argentina de Investigación Clínica (SAIC); 2018

Sociedad Argentina de Investigación Clínica (SAIC)

De novo T3 hormonogenesis requires iodination of Tyr residues and coupling of monoiodo- and diiodo-Tyr within Thyroglobulin (TG) - a 330kDa secretory glycoprotein comprising 4 structural regions including the C-terminal Cholinesterase-Like Domain (ChEL D). Hyperactivation of thyroidal TSH receptors (TSHRs) favor increased de novo T3 formation in TG which is believed secondary to other TG post-translational modifications such as its phosphorylation. Upon TSHR hyperactivation, Fam20C (a secretory serine kinase) is upregulated, accompanied by increased T3 formation. Conversely, suppression of Fam20C decreases de novo T3 synthesis. Interestingly, a phospho-Ser present in hTG occurs at a canonical Fam20C phosphorylation site (equivalent to position S2718 in mTG). We hypothesize that Fam20C-mediated S2718 phosphorylation triggers increased T3 formation in mTG. To analyze the presence of phosphate in the ChEL D, we utilized calf intestinal phosphatase followed by immunoblotting with anti-TG Ab. To study the effects on T3 formation upon disruption of mTG-S2718 and the restitution of the mTG 2718 phospho-environment we bioengineered mTG-S2718A and mTG-S2718E, respectively. We co-expressed the TGs +/- Fam20C, +/- Fam20C inhibitor FL-1607, performed enzymatic iodinations of the secreted TGs, and monitored de novo T3 formation by immunoblotting with mAb anti-T3 and anti-TG Ab. Dephosphorylation of the isolated ChEL D shifts its SDS-PAGE migration and, as we reported, dephosphorylation decreases de novo T3 formation in TG. Co-expression of WT mTG and Fam20C promoted T3 formation in TG and this effect was blocked by Fam20C inhibitor FL-1607. Further, disruption of the established phosphorylation site mTG-S2718A blocked Fam20C-stimulated T3 formation, and this effect was reverted in the phosphomimetic mutant mTG-S2718E. Our data supports the hypothesis that de novo T3 formation in TG is stimulated by Fam20C-mediated phosphorylation of mTG-S2718. Since Fam20C levels are increased in states of TSHR hyperactivation, Fam20C-induced de novo T3 formation may be a contributor to the increased T3 found in hyperstimulated thyroid glands such as occurs in autoimmune hyperthyroidism of Graves´ disease.