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Rewved 19 October I989Four proteins of the hpocortm family, hpocortm I (35 kDa), hpocortm II (36 kDa), lrpocortm V (32 kDa) and hpocortm VI (67-70 kDa), were ldentlfied m the cytosols of 2-day-old cultures of thyroid cells Only hpocortm I was phosphorylated m vitro m fully dlfferentlated, thyroid strmulatmg hormane-treated cells (0 1 mu/ml) Protem kmase C was the only kmase actlvlty which phosphorylated hpocortm I Phosphorylatlon shifted its pI from 6 9 to 6 6 The m v&o phosphorylatlon of hpocorCm I was tmpalred m cultures exposed for 2 days to phorbol ester (IO-' M), although It was present m both the cytosol and the particulate fmctton of these cells Thyroid cells were isolated from ptg giands by ~sconti~uous trypsuuzatlon and cultured as previously described [18] TSH (0 1 mu/ml) or phorbol ester (0 I PM) were added from the start of the culture perrod These protems are Ca"' -pre&ipitabIe and bind to phospho~ipids [2]. They inhibit phospbolipase AZ actrvtty in vrtro and m viva [X%13] and this property decreases after therr pbosphorylation [14]. The exact mechanism of this inhibition remains unknown 1.151.At the end of the Z-day culture period, the cells were washed twice by centrifu~ng at 2oOxg for 7 mm, and the pellet was resuspended m 25 mM E&e-Hepes buffer, pH 7 2 Cells were kept frozen overmght m dry tee before the protan kmase actlvrty was measured Thyroid cell cytosols and partrculate extracts were prepared as described m [17] We recently described the purification and characterization of endonexm (32 kDa) and the presence of lipocortm I (35 kDa) in the pig thyroid gland [16]. We also demonstrated that the major endogenous substrates for thyroid PKC were soIubfe cytosohc proteins with molecular masses between 35 and 38 kDa f 171. Endogenous protem phosphorylatlon m vitroCytosobc and particulate proteins were phosphorylated m vitro with protem kmases A and C as previously described [17] 2 2 1 One-dlmenszonal gel eiectrophoresrs Abquots of mcubation mixtures were subjected to one-dtmenwonaiThe present study provtdes evidence that, among these proteins, Iipocortin I f35 kDa) 1s the best endogenous substrate for the PKC in thyroid stimulating hormone (TSH)-treated Z-day-old cultures of thyroid cells, and that the m vitro phosphorylation of bpocortm I IS Impaired in cultures exposed to phorbol esters for 2 days SDS/polyacrylarnide gel electrophorests (5-15% gel gradients) as described by Laemmh [Is] Iqocortms were identified by Western Blot analysis as described by Tow&n et al [2Oj and mod&ed by Rothhut et al [Zl] The radroactlve phos~hory~at~ protems were vtsualtzed by autora~ography 2 2 2 Two-drmenszonal gel electrophoresrs Samples for two-dimennonal gel electrophoresls were prepared as were those for one-dlmenslonal electrophoresls, except that the concentrations of prott!ms were doubled The phosphoryla~on reactlon was stopped according to @Farrell [22] m the presence of 9 5 M fmai concentration of urea The first clmmenslon separation was by isoelectric focusmg on an amphohne pH gradient from 4 to...

The potent tumor promoter 12-0-tetradecanoyl-phorboll3-acetate (TPA) affects several thyroid cell functions and interacts with thyroid-stimulating hormone (TSH) either by inhibiting or potentiating its action on different cellular parameters. Since phorbol ester acts mainly through the activation of protein kinase C, which is its receptor, we studied this activation and its interaction with TSH and forskolin in suspension cultures of porcine thyroid cells.In thyroid cell cultures, TPA has a dual effect on protein kinase C activity: immediately (2 -5 min) after exposure of cells to TPA, it began to be translocated from the cytosol to the particulate fraction. The transfer of the cytosolic enzyme was total and could occur with or without a loss of activity. The translocated enzyme still needed CaZ+ and phospholipids for its activation. The basal activity increased transiently (2 -4 h) in both the cytosol and particulate fractions during translocation.The peak activity in the particulate fraction was reached 10 -30 min after exposure of cells to TPA, and was followed by down-regulation of protein kinase C and almost complete disappearance of its activity. The residual activity was about 13% of control after a 2-day exposure to TPA. It was unequally distributed between cytosol (4%) and particulate fraction (9%).Prolonged exposure of cells to TPA did not affect either the activity or the subcellular distribution of the CAMP-dependent protein kinase activity. TPA interacted with TSH and prevented the decrease of this activity induced by prolonged exposure of cells to the hormone not only when it was introduced simultaneously with TSH, but also when it was added 24 h after TSH. However, the forskolin-induced decrease in CAMP-dependent protein kinase activity was not prevented by the presence of TPA.TPA also affected the increases in cAMP accumulation mediated by TSH and forskolin. The TSH-induced increase was significantly stimulated by TPA after short contacts ( 5 -15 min), while longer preincubations of cells with TPA provoked a very strong inhibition of the TSH action. However, the forskolin-induced stimulation of the cAMP accumulation was maintained and even further increased in the presence of TPA. Consequently, the actions of TSH and TPA are apparently interdependent, while those of forskolin and TPA seem to be parallel and independent.Neither TSH nor forskolin prevented the TPA-induced down regulation of protein kinase C. The biologically inactive phorbol ester analogue 4a-phorbol 12,13-didecanoate had no effect on protein kinase C activity, and did not interact with either TSH or forskolin.Endogenous substrates of the two protein kinases in cultured thyroid cells were revealed by gel electrophoresis after phosphorylation of cytosolic proteins by these enzymes. Three protein bands (28, 30 and 33 kDa) were specifically phosphorylated by CAMP-dependent protein kinase; the molecular masses of the proteins phosphorylated by protein kinase C were 35 -38 kDa.There was a close parallel between the two protein kinase a...

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