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MOLECULAR MECHANISMS UNDERLYING INCREASED DE NOVO TRIIODOTHYRONINE FORMATION WITHIN THYROGLOBULIN IN HYPERSTIMULATED THYROCYTES
IBAÑEZ PADILLA, CAROLINA; ARÉVALO, EZEQUIEL; ARVAN, PETER; TARGOVNIK , HÉCTOR M; CITTERIO, CINTIA E
Mar del PLata
Congreso; LXIII Reunión Científica Anual de la Sociedad Argentina de Investigación Clínica; 2018
Sociedad Argentina de Investigación Clínica
Thyroglobulin (TG), a 330kDa secretory glycoprotein comprising 4 structural regions, is the scaffold for de novo triiodothyronine (T3) formation which requires iodination and coupling of two iodotyrosines within TG. Hyperactivation of thyroidal TSH receptors (TSHRs) favor increased de novo T3 formation in TG as a consequence of other TG post-translational modifications such as its phosphorylation. Fam20C (a secretory serine kinase) is upregulated by TSHR hyperstimulation, accompanied by increased T3 formation in TG. 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 study the effects on T3 formation upon disruption of mTG-S2718 and the restitution of the mTG 2718 phospho-environment we bioengineered mTGS2718A and mTG-S2718E, respectively. We co-expressed the TGs +/- Fam20C, +/- Fam20C inhibitor FL-1607 in 293T cells (lacking endogenous TG expression and with negligible endogenous Fam20C expression), performed enzymatic iodinations of the secreted TGs, and monitored de novo T3 formation by immunoblotting with mAb anti-T3 and polyclonal anti-TG Ab. 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.