Characterization and identification as cofilin and destrin of two thyrotropin- and phorbol ester-regulated phosphoproteins in thyroid cells

Abstract

Using separation of total cellular proteins by two dimensional (2-D) gel electrophoresis (isoelectric focusing/SDS-PAGE) we have characterized 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-Prot protein sequence database revealed that p19 was identical to destrin/ADF (actin depolymerizing factor) and p21 to cofilin, two closely related and widely distributed actin-binding proteins. This was further verified by cross-reactivity with specific antibodies against brain cofilin and chicken ADF. We have demonstrated, using 2-D gel electrophoresis with a nonequilibrium pH gradient in the first dimension (nonequilibrium pH gradient in the first dimension (nonequilibrium pH gradient electrophoresis/SDS-PAGE) that, in the thyroid cell, cofilin and destrin/ADF were present, under control conditions, in two forms: a phosphorylated and an unphosphorylated one. Thyrotropin (TSH), through cyclic AMP, provoked a very rapid dephosphorylation of these two proteins, which was already maximal after 20 min of action, whereas 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 of actin-containing stress fibers and in the reorganization of microfilaments induced by this hormone. Epidermal growth factor, which does not induce acute morphological changes in thyroid cells, did not affect the state of phosphorylation of cofilin and destrin/ADF except for a delayed decrease (after 24 h) of destrin/ADF phosphorylation. A 10% dimethyl sulfoxide treatment of thyroid cells also induced rapid dephosphorylation of destrin and cofilin. This was accompanied by a reorganization of actin microfilaments that clearly resembles the one induced by TSH and by the appearance of intranuclear cofilin-containing rods. However, these rod structures were not observed in response to TSH, forskolin, or TPA, suggesting that dephosphorylation of cofilin correlates with the reorganization of actin microfilaments but not with the nuclear transport of cofilin. We propose that the dephosphorylation of destrin and cofilin could be involved in the TSH-stimulated macropinocytic activity, a key process in thyroid hormone secretion.