In vitro protein kinase C phosphorylation sites of placental lipocortin

Schlaepfer, David D.; Haigler, Harry T.

doi:10.1021/bi00412a008

Cited by 79 publications

(56 citation statements)

References 36 publications

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“…Human lipocortin 1 has been shown to be a substrate for epidermal growth factor receptor kinase (Pepinski & Sinclair, 1986;Haigler et al, 1987;Futter et al, 1993) and protein kinase C (Varticovski et al, 1988;Schlaepfer & Haigler, 1988;Oudinet et al, 1993). Lipocortin 1 has also been implicated in the regulation of signal transduction pathways involving phospholipase A2 inhibition (Kim et al, 1994).…”

Section: Introductionmentioning

confidence: 99%

Translocation of lipocortin (annexin) 1 to the membrane of U937 cells induced by phorbol ester, but not by dexamethasone

Kang

Cho

Moon

et al. 1996

British J Pharmacology

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1 Induction of lipocortin 1 secretion by dexamethasone has been demonstrated, although the secretory mechanism is still unknown. We have studied the effects of 12-tetradecanoyl phorbol 13-acetate (TPA) and/or dexamethasone on the expression, translocation, and secretion of lipocortin 1 in U937 cells. 2 The expression of lipocortin 1 and its mRNA increased during TPA-induced differentiation of U937 cells to a maximum of 1.9 fold and 8.2 fold, respectively, after 48 h. Both the protein and the mRNA levels decreased after 48 h. 3 TPA caused the translocation of lipocortin 1 from the cytosol to the membrane of U937 cells in a time-dependent manner, as determined by Western blot analysis. The translocation was concurrent with the differentiation of the cells. After 48 h of TPA treatment, 82.6+6.5% of lipocortin 1 was present in the membrane fraction compared to 41.6+1.7% in untreated cells. 4 The amount of lipocortin 1 that was externally bound (associated) with the membrane increased to 3.2 fold as the cytosol to membrane translocation of lipocortin 1 increased. 5 Dexamethasone decreased the externally bound lipocortin 1, but had no effect on the cytosol to membrane translocation. 6 This offers a model system with which the function and the secretion mechanism of lipocortin 1 can be studied. Our data is consistent with the hypothesis that the secretory mechanism is through an unknown pathway, involving the translocation of lipocortin 1 from the cytosol to the internal membranes, and then, its secretion to the external membrane.

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Section: Introductionmentioning

confidence: 99%

Translocation of lipocortin (annexin) 1 to the membrane of U937 cells induced by phorbol ester, but not by dexamethasone

Kang

Cho

Moon

et al. 1996

British J Pharmacology

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“…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.…”

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confidence: 99%

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

Rothhut

Dubois

Féliers

et al. 1995

European Journal of Biochemistry

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U 96 INSERM, Le Kremlin-Bicetre, FranceAnnexin V belongs to a large family of calcium-binding and phospholipid-binding proteins and may act as an endogenous regulator of the protein kinase C (PKC) activity. This study examines the effect of annexin V on the in v i m PKC activity in cultured mesangial cells using histone H1, the peptide [Ser25]PKC-(19-31), or endogenous proteins as substrates. The SDS/PAGE pattern of "P-labeled mesangial proteins showed that the calcium-independent PKC [(n+a)PKC] phosphorylated several proteins from 70 kDa to 40 kDa and 22 kDa to 15 kDa. Three additional proteins from 34 kDa to 29 kDa, including annexin I and its proteolytic forms, were detected after activation of calcium-dependent PKC (cPKC). Increasing concentrations of annexin V did not alter the phosphorylation of (n+a)PKC substrates. By contrast, specific phosphorylation of proteins and annexin I by cPKC, was reduced in a dose-dependent manner. Addition of high concentration of calcium and phosphatidylserine did not reverse the inhibitory effect of annexin V. Annexin V also inhibited the phosphorylation of histone HI or peptide [Ser25]PKC-(19-31) by cPKC. Moreover, removal of annexin V from cytosols increased the annexin I phosphorylation by these isoforms. From these results, we propose that annexin V may regulate the signal-transduction pathway involving the activation of cPKC, as they act in vitro as an inhibitor of these kinases.

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“…The pi of the phosphorylated form of lipocortin I was different (66) from that of the non-phosphorylated molecules (6,9), This may indicate either that all molecules were not phosphorylated under our experimental conditions (3 min) or that the lipocortin I molecule population is heterogeneous. The latter could explain the lack of phosphorylation of particulate lipocortins.…”

Section: Discussionmentioning

confidence: 79%

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

Antonicelli¹,

Omri

Breton

et al. 1989

FEBS Letters

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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...

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In vitro protein kinase C phosphorylation sites of placental lipocortin

Cited by 79 publications

References 36 publications

Translocation of lipocortin (annexin) 1 to the membrane of U937 cells induced by phorbol ester, but not by dexamethasone

Translocation of lipocortin (annexin) 1 to the membrane of U937 cells induced by phorbol ester, but not by dexamethasone

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

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

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