EVIDENCE THAT THYROGLOBULIN HAS AN ASSOCIATED PROTEIN-KINASE ACTIVITYCORRELATED WITH THE PRESENCE OF AN ADENOSINE-TRIPHOSPHATE BINDING-SITE

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 FRTL(5) rat thyroid cells. Subject ed to rigorous purification procedures, all Tg preparations showed a p rotein kinase activity that is able to phosphorylate serine residues i n 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 phosp hotransferase activity in all Tg preparations tested. Furthermore, Tg contains a specific and saturable ATP binding site, as evidenced by sp ecific binding with the ATP affinity analog p-fluorosulfonylbenzoyl 5' -p-adenosine (FSO(2)BzAdo). Limited proteolysis of FSO2[C-14]BzAdo-lab eled human goiter Tg with clostripain gave rise to a M(r) 64,000 amino terminal polypeptide carrying almost all of the label. The analysis o f this fragment revealed two sequences that may play an analogous phos phate-loop (P-loop) function, even though their primary sequences diff er slightly from the classical A consensus sequence or P-loop. The ATP binding site of Tg may be functionally associated with the presence o f a protein kinase A phosphorylating activity linked to the Tg itself, because the addition of the analog is able to inhibit the enzymatic a ctivity in a dose-dependent fashion. Contamination with non-Tg phospho rylase cannot be ruled out at present, even though the amount of ATP t hat Tg was able to bind did not significantly change during the variou s purification steps. Together these findings favor the hypothesis tha t the Tg molecule contains a protein kinase activity that is capable o f autophosphorylation.