CHARACTERIZATION AND IDENTIFICATION AS COFILIN AND DESTRIN OF 2 THYROTROPIN-REGULATED AND PHORBOL ESTER-REGULATED PHOSPHOPROTEINS IN THYROID-CELLS

Using separation of total cellular proteins by two dimensional (2-D) g el electrophoresis (isoelectric focusing/SDS-PAGE) we have characteriz ed two regulated proteins, p21 and p19, in dog thyroid cells. We have used the same 2-D gel technique to purify these proteins before their trypsin cleavage and partial sequencing. Three peptides were sequenced in the case of p19 and two peptides in the case of p21. The Swiss-Pro t protein sequence database revealed that p19 was identical to destrin /ADF (actin depolymerizing factor) and p21 to cofilin, two closely rel ated and widely distributed actin-binding proteins. This was further v erified by crossreactivity with specific antibodies against brain cofi lin and chicken ADF. We have demonstrated, using 2-D gel electrophores is with a nonequilibrium pH gradient in the first dimension (nonequili brium pH gradient electrophoresis/SDS-PAGE) that, in the thyroid cell, cofilin and destrin/ADF were present, under control conditions, in tw o forms: a phosphorylated and an unphosphorylated one. Thyrotropin (TS H), through cyclic AMP, provoked a very rapid dephosphorylation of the se two proteins, which was already maximal after 20 min of action, whe reas their dephosphorylation in response to 12-O-tetradecanoylphorbol- 13-acetate (TPA) was slower. This suggests that dephosphorylation of cofilin and destrin/ADF by TSH could be implicated in the disruption o f actin-containing stress fibers and in the reorganization of microfil aments induced by this hormone. Epidermal growth factor, which does no t induce acute morphological changes in thyroid cells, did not affect the state of phosphorylation of cofilin and destrin/ADF except for a d elayed decrease (after 24 h) of destrin/ADF phosphorylation. A 10% dim ethyl sulfoxide treatment of thyroid cells also induced rapid dephosph orylation of destrin and cofilin. This was accompanied by a reorganiza tion of actin microfilaments that clearly resembles the one induced by TSH and by the appearance of intranuclear cofilin-containing rods. Ho wever, these rod structures were not observed in response to TSH, fors kolin, or TPA, suggesting that dephosphorylation of cofilin correlates with the reorganization of actin microfilaments but not with the nucl ear transport of cofilin. We propose that the dephosphorylation of des trin and cofilin could be involved in the TSH-stimulated macropinocyti c activity, a key process in thyroid hormone secretion. (C) 1994 Acade mic Press, Inc.