T. Saito et al., CHARACTERIZATION AND IDENTIFICATION AS COFILIN AND DESTRIN OF 2 THYROTROPIN-REGULATED AND PHORBOL ESTER-REGULATED PHOSPHOPROTEINS IN THYROID-CELLS, Experimental cell research, 212(1), 1994, pp. 49-61
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.