Endocrinology, Vol 136, 3179-3185, Copyright © 1995 by Endocrine Society

ARTICLES

Evidence that thyroglobulin has an associated protein kinase activity correlated with the presence of an adenosine triphosphate binding site

CG Alvino, AM Acquaviva, AM Catanzano and V Tassi
Centro di Endocrinologia ed Oncologia Sperimentale del Consiglio Nazionale delle Ricerche, Naples, Italy.

19S Thyroglobulin (Tg), a dimeric glycoprotein with an M(r) of 660,000, was extracted from rat, bovine, and human (goitrous) thyroid tissues, as well as from culture medium of FRTL5 rat thyroid cells. Subjected to rigorous purification procedures, all Tg preparations showed a protein kinase activity that is able to phosphorylate serine residues in vitro. Further characterization of this enzymatic activity revealed that Tg has the specificities of a protein kinase A when Kemptide was used as specific substrate and after analysis of cAMP-stimulated phosphotransferase activity in all Tg preparations tested. Furthermore, Tg contains a specific and saturable ATP binding site, as evidenced by specific binding with the ATP affinity analog p-fluorosulfonylbenzoyl 5'-p-adenosine (FSO2BzAdo). Limited proteolysis of FSO2[14C]BzAdo- labeled human goiter Tg with clostripain gave rise to a M(r) 64,000 amino terminal polypeptide carrying almost all of the label. The analysis of this fragment revealed two sequences that may play an analogous phosphate-loop (P-loop) function, even though their primary sequences differ slightly from the classical A consensus sequence or P- loop. The ATP binding site of Tg may be functionally associated with the presence of a protein kinase A phosphorylating activity linked to the Tg itself, because the addition of the analog is able to inhibit the enzymatic activity in a dose-dependent fashion. Contamination with non-Tg phosphorylase cannot be ruled out at present, even though the amount of ATP that Tg was able to bind did not significantly change during the various purification steps. Together these findings favor the hypothesis that the Tg molecule contains a protein kinase activity that is capable of autophosphorylation.

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