Identification of four lipocortin proteins and phosphorylation of lipocortin I by protein kinase C in cytosols of porcine thyroid cell cultures

Abstract

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...

Characterization of the Annexin I Gene and Evaluation of Its Role in Type 2 Diabetes

“…Human ANXA1 (also called calpactin II, lipocortin 1, or p35) belongs to the annexin superfamily, which consists of at least 11 different, abundant, and ubiquitously expressed proteins (6). In vitro studies have suggested that ANXA1 may play an important role in the regulation of both insulin secretion (6 -12), including stimulation of insulin secretion by the arachidonic acid-phospholipase A 2 (cPLA 2 ) pathway (13,14) or in the signal cascades initiated from the insulin receptor (7,8).…”

Inhibitory Effect of Annexin V on Protein Kinase C Activity in Mesangial Cell Lysates

“…Activation of PKC by growth factors, hormones or phorbol esters leads to the phosphorylation of many endogenous proteins which mediate the effects of these agonists on cellular processes such as proliferation and differentiation. Annexins are among these substrates phosphorylated by PKC (Khanna et al, 1986;Schlaepfer and Haigler, 1988;Antonicelli et al, 1989;Isacke et al, 1986;Could et al, 2986). In mesangial cells, we have shown that actiCrmespondence to J.-P. Oudinet PtdIns(4,5)P2, phosphatidylinositol 4,s-bisphosphate; Ins(l,4,5)P3, inositol 1,4,5-trisphosphate; acyl,Gro, diacylglycerol; PKC, protein kinase C; c-, conventional; n-, novel; a-, atypical.…”

Production of lipocortin‐like proteins by cultured human tracheal submucosal gland cells

“…After permeabilization, the cell membrane was clearly stained (Fig. 1B) [31], cultured rat renomedullary interstitial cells [4], porcine thyroid cells [32], human endothelial cells [33] and human mononuclear cells [34]. Several reports have shown that the 35 kDa and 67-70 kDa lipocortin-like protein are closely related with respect to sequence, antiserum cross-reactivity and binding of Ca 2+ and phospholipid (see [35] for refs).…”

“…Lipocortin I is a substrate for both the EGF receptor tyrosyl kinase [36] and the protein kinase C (PKC) [2,37]. Antonicelli et al [32] recently showed that among four proteins (32, 35, 36 and 67-70 kDa) of the lipocortin family, only lipocortin I was the endogenous substrate for the PKC in thyroid-stimulating hormone (TSH) treated cells. This is the first evidence that lipocortin-like proteins can be produced and released by human airway submucosal secretory cells.…”