Cellular and molecular characterization of IDH1-mutated diffuse low grade gliomas reveals tumor heterogeneity and absence of EGFR/PDGFRα activation.

Diffuse low grade gliomas (DLGG, grade II gliomas) are slowly-growing brain tumors that often progress into high grade gliomas. Most tumors have a missense mutation for IDH1 combined with 1p19q codeletion in oligodendrogliomas or ATRX/TP53 mutations in astrocytomas. The phenotype of tumoral cells, their environment and the pathways activated in these tumors are still ill-defined and are mainly based on genomics and transcriptomics analysis. Here we used freshly-resected tumors to accurately characterize the tumoral cell population and their environment. In oligodendrogliomas, cells express the transcription factors MYT1, Nkx2.2, Olig1, Olig2, Sox8, four receptors (EGFR, PDGFRα, LIFR, PTPRZ1) but not the co-receptor NG2 known to be expressed by oligodendrocyte progenitor cells. A variable fraction of cells also express the more mature oligodendrocytic markers NOGO-A and MAG. DLGG cells are also stained for the young-neuron marker doublecortin (Dcx) which is also observed in oligodendrocytic cells in nontumoral human brain. In astrocytomas, MYT1, PDGFRα, PTPRZ1 were less expressed whereas Sox9 was prominent over Sox8. The phenotype of DLGG cells is overall maintained in culture. Phospho-array screening showed the absence of EGFR and PDGFRα phosphorylation in DLGG but revealed the strong activation of p44/42 MAPK/ERK which was present in a fraction of tumoral cells but also in nontumoral cells. These results provide evidence for the existence of close relationships between the cellular phenotype and the mutations found in DLGG. The slow proliferation of these tumors may be associated with the absence of activation of PDGFRα/EGFR receptors.

Elastokine-mediated up-regulation of MT1-MMP is triggered by nitric oxide in endothelial cells.

Membrane-type I matrix metalloproteinase (MT1-MMP) has been previously reported to be up-regulated in human microvascular endothelial cell-1 line (HMEC) by elastin-derived peptides (elastokines). The aim of the present study was to identify the signaling pathways responsible for this effect. We showed that elastokines such as (VGVAPG)(3) peptide and kappa elastin induced nitric oxide (NO) production in a time-, concentration- and receptor-dependent manner as it could be abolished by lactose and a receptor-derived competitive peptide. As evidenced by the use of NO synthase inhibitors, elastokine-mediated up-regulation of MT1-MMP and pseudotube formation on Matrigel required NO production through activation of the PI(3)-kinase/Akt/NO synthase and NO/cGMP/Erk1/2 pathways. Elastokines induced both PI(3)-kinase p110gamma sub-unit, Akt and Erk1/2 activation, as shown by a transient increase in phospho-Akt and phospho-Erk1/2, reaching a maximum after 5 and 15 min incubation, respectively. Inhibitors of PI(3)-kinase and MEK1/2 suppressed elastokine-mediated MT1-MMP expression at both the mRNA and protein levels, and decreased the ability of elastokines to accelerate pseudotube formation. Besides, elastokines mediated a time- and concentration-dependent increase of cGMP, suggesting a link between NO and MT1-MMP expression. This was validated by the use of a guanylyl cyclase inhibitor, a NO donor and a cGMP analog. The guanylyl cyclase inhibitor abolished the stimulatory effect of elastokines on MT1-MMP expression. Inversely, the cGMP analog, mimicked the effect of both elastokines and NO donor in a concentration- and time-dependent manner. Overall, our results demonstrated that such elastokine properties through NO and MT1-MMP may be of importance in the context of tumour progression.

The C-terminal CD47/IAP-binding domain of thrombospondin-1 prevents camptothecin- and doxorubicin-induced apoptosis in human thyroid carcinoma cells.

Camptothecin and doxorubicin belong to a family of anticancer drugs that exert cytotoxic effects by triggering apoptosis in various cell types. However there have only been few investigations showing that matricellular proteins like thrombospondin-1 (TSP-1) could be involved in the underlying mechanism of this cytotoxicity. In this report, using Hoechst reagent staining, reactive oxygen species production and caspase-3 activity measurement, we determined that both camptothecin and doxorubicin induced apoptosis in human thyroid carcinoma cells (FTC-133). On the one hand, we demonstrated that camptothecin and doxorubicin inhibited TSP-1 expression mainly occurring at the transcriptional level. On the other hand, drug-induced apoptosis determined by western blot analysis for PARP cleavage and caspase-3 activity measurement, was significantly decreased in presence of exogenous TSP-1. In order to identify the sequence responsible for this effect, we used the CD47/IAP-binding peptide 4N1 (RFYVVMWK), derived from the C-terminal domain of TSP-1, and known to play a role in apoptosis. Thus, in presence of 4N1, camptothecin and doxorubicin-induced pro-apoptotic activity was considerably inhibited. These findings suggest that induction of apoptosis by camptothecin or doxorubicin in FTC-133 cells is greatly dependent by a down-regulation of TSP-1 expression and shed new light on a possible role for TSP-1 in drug resistance.

Angiotensin II-induced transcriptional activation of the cyclin D1 gene is mediated by Egr-1 in CHO-AT(1A) cells.

Cyclin D1 protein expression is regulated by mitogenic stimuli and is a critical component in the regulation of G(1) to S phase progression of the cell cycle. Angiotensin II (Ang II) binds to specific G protein-coupled receptors and is mitogenic in Chinese hamster ovary cells stably expressing the rat vascular Ang II type 1A receptor (CHO-AT(1A)). We recently reported that in these cells, Ang II induced cyclin D1 promoter activation and protein expression in a phosphatidylinositol 3-kinase (PI3K)-, SHP-2-, and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK)-dependent manner (Guillemot, L., Levy, A., Zhao, Z. J., Béréziat, G., and Rothhut, B. (2000) J. Biol. Chem. 275, 26349-26358). In this report, transfection studies using a series of deleted cyclin D1 promoters revealed that two regions between base pairs (bp) -136 and -96 and between bp -29 and +139 of the human cyclin D1 promoter contained regulatory elements required for Ang II-mediated induction. Mutational analysis in the -136 to -96 bp region provided evidence that a Sp1/early growth response protein (Egr) motif was responsible for cyclin D1 promoter activation by Ang II. Gel shift and supershift studies showed that Ang II-induced Egr-1 binding involved de novo protein synthesis and correlated well with Egr-1 promoter activation. Both U0126 (an inhibitor of the MAPK/ERK kinase MEK) and wortmannin (an inhibitor of PI3K) abrogated Egr-1 endogenous expression and Egr-1 promoter activity induced by Ang II. Moreover, using a co-transfection approach, we found that Ang II induction of Egr-1 promoter activity was blocked by dominant-negative p21(ras), Raf-1, and tyrosine phosphatase SHP-2 mutants. Identical effects were obtained when inhibitors and dominant negative mutants were tested on the -29 to +139 bp region of the cyclin D1 promoter. Taken together, these findings demonstrate that Ang II-induced cyclin D1 up-regulation is mediated by the activation and specific interaction of Egr-1 with the -136 to -96 bp region of the cyclin D1 promoter and by activation of the -29 to +139 bp region, both in a p21(ras)/Raf-1/MEK/ERK-dependent manner, and also involves PI3K and SHP-2.

The protein-tyrosine phosphatase SHP-2 is required during angiotensin II-mediated activation of cyclin D1 promoter in CHO-AT1A cells.

Angiotensin II (Ang II) binds to specific G protein-coupled receptors and is mitogenic in Chinese hamster ovary (CHO) cells stably expressing a rat vascular angiotensin II type 1A receptor (CHO-AT(1A)). Cyclin D1 protein expression is regulated by mitogens, and its assembly with the cyclin-dependent kinases induces phosphorylation of the retinoblastoma protein pRb, a critical step in G(1) to S phase cell cycle progression contributing to the proliferative responses. In the present study, we found that in CHO-AT(1A) cells, Ang II induced a rapid and reversible tyrosine phosphorylation of various intracellular proteins including the protein-tyrosine phosphatase SHP-2. Ang II also induced cyclin D1 protein expression in a phosphatidylinositol 3-kinase and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK)-dependent manner. Using a pharmacological and a co-transfection approach, we found that p21(ras), Raf-1, phosphatidylinositol 3-kinase and also the catalytic activity of SHP-2 and its Src homology 2 domains are required for cyclin D1 promoter/reporter gene activation by Ang II through the regulation of MAPK/ERK activity. Our findings suggest for the first time that SHP-2 could play an important role in the regulation of a gene involved in the control of cell cycle progression resulting from stimulation of a G protein-coupled receptor independently of epidermal growth factor receptor transactivation.

Participation of annexins in protein phosphorylation.

Simultaneous discovery of members of the annexin family of calcium and phospholipid binding proteins by several groups is intimately linked to the possibility that these proteins may be controlled by phosphorylation. Indeed, annexin I and annexin II have been identified as major substrates for the tyrosine kinase activity associated with epidermal growth factor receptor (EGF-R) and for the retrovirus encoded protein tyrosine kinase pp60v-arc. Both annexins are also in vitro and/or in situ substrates for platelet derived growth factor (PDGF), insulin and hepatocyte growth factor/scatter factor (HGF/SF) receptor tyrosine kinases. In addition, to serve as substrates for tyrosine protein kinases some annexins are cellular targets for serine threonine protein kinases such as protein kinase C (PKC) and cAMP-dependent protein kinase A (PKA). Although the role of annexin phosphorylation has not been studied in detail, it is thought to influence their vesicle aggregation and phospholipid binding properties. Some annexins are also potent inhibitors of various serine/threonine and tyrosine kinases. The physiological functions of the annexins have still not been clearly defined. Therefore the identification of the ability of these proteins to undergo phosphorylation may be helpful in assigning them a precise biological role.

Post-translational regulation of neuronal acetylcholine receptors stably expressed in a mouse fibroblast cell line.

Second messenger regulation of neuronal acetylcholine receptors (AChRs) was investigated in a mouse fibroblast cell line, M10, stably transfected with chicken alpha 4 and beta 2 cDNAs. Both forskolin and 8-bromo-cyclic adenosine 3',5'-monophosphate (cAMP) induced large increases in the numbers of AChRs. The increases were due in part to increased transcription and translation of the alpha 4 and beta 2 genes. Blockade of protein synthesis with cycloheximide, however, revealed that forskolin also exerts a post-translational effect, increasing the number of surface receptors by twofold. Immunoblot analysis of sucrose gradient fractions confirmed that the cells had a large fraction of unassembled subunits potentially available for receptor assembly. The post-translational effect of forskolin was blocked by H-89, an inhibitor of cAMP-dependent protein kinase, and by okadaic acid, an inhibitor of phosphatases 1 and 2A. Nicotine also acted post-translationally to induce a twofold increase in the number of surface receptors, but the mechanism differed from that utilized by forskolin, since the effects of the two agents were additive and were differentially affected by okadaic acid. The results suggest that protein phosphorylation-dephosphorylation mechanisms act post-translationally to increase the number of neuronal AChRs maintained on the cell surface. This could be achieved by increasing the efficiency of receptor assembly, transport, or stabilization on the cell surface.

Inhibitory effect of annexin V on protein kinase C activity in mesangial cell lysates.

Annexin 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 vitro 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 32P-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 H1 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.

In vivo and in vitro phosphorylation of annexin II in T cells: potential regulation by annexin V.

In order to understand how signal transduction occurs during T cell activation, it is necessary to identify the key regulatory molecules whose function is influenced by phosphorylation. Annexins II (A-II) and V (A-V) belong to a large family of Ca(2+)-dependent phospholipid-binding proteins. Among many putative functions, annexins may be involved in signal transduction during cellular proliferation and differentiation. In the present study we show that A-II is phosphorylated in vivo in the Jurkat human T cell line. Indeed, A-II is phosphorylated after stimulation by phorbol myristate acetate and on serine residues after T cell antigen receptor (TcR) stimulation. In cytosol from Jurkat cells, A-II is phosphorylated only by Ca2+/phospholipid-stimulated kinases such as Ca(2+)-dependent protein kinases C (cPKCs). A-V inhibits the phosphorylation of A-II and other substrates of cPKCs and has no effect on kinases activated only by phospholipids. In conclusion, A-II is phosphorylated both in vitro and in vivo in Jurkat cells, and may play a role as a substrate during signal transduction in lymphocytes via the TcR through the PKC pathway. On the other hand, A-V could act as a potent modulator of cPKCs in Jurkat cells.

High levels of antibodies to annexins V and VI in patients with rheumatoid arthritis.

OBJECTIVE: Glucocorticoids are powerful antiinflammatory agents widely used for the treatment of rheumatoid arthritis (RA). Synthesis and/or secretion of annexin I (A-I) is induced by these steroids. Annexins V and VI are also found extracellularly but are not induced by glucocorticoids. Annexins may be potent antagonists of phospholipase A2 (PLA2). Since autoantibodies to A-I have been reported in patients with RA, we studied the reactivity of sera from patients with RA to A-V and A-VI. METHODS: Sera from 26 patients with RA were assessed for anti-A-V and anti-A-VI antibodies and compared with sera from 26 sex/age matched healthy subjects. IgG and IgM antibodies were analyzed in an ELISA: A correlation study with disease activity and corticosteroid treatment schedule was performed. RESULTS: Sera from patients with RA contained significantly higher levels of IgG [anti-A-V and anti-A-VI] autoantibodies than control sera, both being correlated. This rise in antiannexin antibody titers was correlated with the RA activity score, and negatively correlated with the daily dose of corticosteroids. CONCLUSION: High levels of IgG (anti-A-V and anti-A-VI) antibodies were found in sera from patients with RA. We suggest that antiannexin autoantibodies may play a role in the clinical course of RA by impairing the anti-PLA2 effect of annexins.

Potential interaction between annexin VI and a 56-kDa protein kinase in T cells.

Annexins belong to a large family of calcium-dependent phospholipid binding proteins known to undergo post-translational modifications such as phosphorylation. Physiological function of each annexin is still unclear since they may participate in signal transduction. We have tested the presence of annexins in a T cell line (Jurkat) and studied their phosphorylation by protein tyrosine kinases of the src family. Among annexins I, II, V and VI found in Jurkat cells, annexin VI was shown to be phosphorylated in vitro by p56lck and annexins I and II by p60src. We could not detect the phosphorylation of A-VI in vivo, even after cell stimulation. However, a 56-kDa phosphoprotein was found to be associated with A-VI after T cell activation. This 56-kDa protein shares some characteristics with p56lck.

Anti-CD3 and phorbol ester induce distinct phosphorylated sites in the SH2 domain of p56lck.

P56lck is a protein tyrosine kinase of the Src family specifically expressed in T lymphocytes. Triggering of T cells with anti-CD3 or with phorbol 12-myristate 13-acetate (PMA) results in the appearance of slower migrating forms (shift) of p56lck. To investigate the phosphorylation sites on the shifted forms of p56lck and to assess the role of protein kinase C in this phosphorylation, Jurkat cells were treated with a selective inhibitor of this kinase (GF 109203X). This inhibitor completely reversed the shift induced by PMA but only partially reversed the one induced after triggering with anti-CD3. To analyze the shift further, p56lck was immunoprecipitated from in vivo labeled cells treated either with anti-CD3 or with PMA. Tryptic phosphopeptides were generated and analyzed by using a combination of thin layer chromatography, high reticulation polyacrylamide gel electrophoresis, reverse phase chromatography, and phosphopeptide sequencing. We identified serine 158 as a newly phosphorylated site after PMA treatment and tyrosine 192 and serine 194 in the major tryptic phosphopeptide obtained after anti-CD3 triggering. The three sites identified are located in the SH2 domain of p56lck; this suggests that their phosphorylation may regulate the interaction with other proteins or with other internal domains in p56lck.

Expression of annexin I, II, V, and VI by rat osteoblasts in primary culture: stimulation of annexin I expression by dexamethasone.

To determine whether rat osteoblasts synthesize proteins of the annexin family and to evaluate the extent to which glucocorticoids modulate the expression of annexins by these cells, osteoblasts were grown in primary cultures in the absence or presence of dexamethasone, and the expression of annexins was evaluated by immunoblotting using polyclonal antibodies against human annexins. Four different annexins (I, II, V, and VI) were found to be expressed by rat osteoblasts. The expression of annexin I, but not the other annexins studied, was increased in osteoblasts cultured in the presence of dexamethasone (173 +/- 33% increase comparing untreated cells and cells treated for 10 days with 5 x 10(-7) M dexamethasone). Increased expression of annexin I was observed after the third day of exposure to dexamethasone and rose thereafter until day 10; annexin I expression increased with dexamethasone concentrations above 10(-10) M throughout the range of concentrations studied. The increase in annexin I protein was associated with an increase in annexin I mRNA and was completely blocked by the concomitant addition of the glucocorticoid receptor antagonist RU 38486. The increase in annexin I content following dexamethasone treatment was associated with an increase in alkaline phosphatase activity and PTH-induced cAMP stimulation, whereas phospholipase A2 activity in the culture medium was reduced to undetectable levels. The finding that four annexins are expressed in rat osteoblasts in primary culture raises the possibility that these proteins could play an important role in bone formation by virtue of their ability to bind calcium and phospholipids, serve as Ca2+ channels, interact with cytoskeletal elements, and/or regulate phospholipase A2 activity. In addition, the dexamethasone-induced increase in annexin I may represent a mechanism by which glucocorticoids modify osteoblast function.

Annexins from bovine adrenal cortex exhibit specific cytosol/membrane solvation.

Cytosol/membrane localization of annexins I to VI was analyzed in tissue extracts from bovine adrenal cortex. Based on their solubility in either aqueous or detergents solutions, they were subfractionated in three groups named cytosolic (C), membrane-bound (MB) and membrane-inserted (MI). Less than 1% of the total annexins present in the tissue were recovered in the C fraction when as much as 76.5 and 22.5% were obtained respectively in the MB and the MI fractions. By immunoblotting after SDS-PAGE, it was shown that the various members of the annexin family were not equally recovered in the different fractions. A-V and A-VI were found present in the three fractions whereas the distribution of A-I, A-II, A-III and A-IV was distinct, suggesting different cellular functions.

Protein kinase C-dependent phosphorylation of annexins I and II in mesangial cells.

In this study we describe the phosphorylation of annexins from cultured rat mesangial cells by protein kinase C (PKC) both in vitro and in vivo. Annexins I and II were detected either by Western-blot analysis or by immunoprecipitation using specific antibodies. In the presence of [gamma-32P]ATP, cytosolic annexin I and annexin II were phosphorylated in vitro only when Ca2+ and phospholipids were added, but not in the presence of phospholipids alone. Annexin I was shown to be a better substrate than annexin II. In experiments in vivo performed on 32P-labelled mesangial cells, the addition of two well-known activators of PKC, namely angiotensin II (AII) and phorbol myristate acetate (PMA), increased preferentially the phosphorylation of annexin I. Annexin II was phosphorylated to a much lesser extent after AII treatment. Phosphoamino acid analysis of annexins, either by two-dimensional chromatography or by using a specific antiphosphotyrosine antibody, revealed only phosphoserine in these experiments in vivo. The addition of AII to mesangial cells increased serine phosphorylation of annexin I and annexin II, whereas PMA only increased serine phosphorylation of annexin I. V8-protease phosphopeptide mapping of annexin I that was phosphorylated both in vitro and in vivo by PKC from mesangial cells shows similar phosphopeptides.

Recombinant Csk expressed in Escherichia coli is autophosphorylated on tyrosine residue(s).

The C-terminal src kinase (Csk) is responsible for the phosphorylation of the carboxy-terminal tyrosine of several tyrosine kinases of the Src family. This phosphorylation site has a negative regulatory function. Csk is unique among the members of the protein tyrosine kinase family because it lacks the conserved tyrosine autophosphorylation site and has been thought to be devoid of autophosphorylation activity. Using the glutathione S-transferase (GST) bacterial expression system, we have produced large amounts of a chimeric rat Csk protein. We have determined that the GST-Csk fusion protein isolated from bacteria is autophosphorylated on tyrosine residue(s). GST-Csk and purified Csk are capable of undergoing autophosphorylation on tyrosine residue(s) in vitro. The GST-Csk fusion protein also phosphorylates exogenous substrates, including the heteropolymer poly-Glu/Tyr and enolase. This is the first indication that Csk is autophosphorylated on tyrosine residue(s) both in vivo in bacteria expressing Csk cDNA and in vitro. These findings suggest that the autophosphorylation of Csk might play a role in the regulation of its kinase activity as well as its binding to other cellular proteins.

The lymphocyte-specific tyrosine protein kinase p56lck is endocytosed in Jurkat cells stimulated via CD2.

Incubation of the human T cells, Jurkat, with two sets of activating anti-CD2 mAb (T11(2) + T11(3), D66 + T11(1)) induced delocalization of p56lck and CD2 receptors from the plasma membrane and increased the tyrosine kinase activity of p56lck. The anti-CD2 mAb combination (T11(2) + T11(3)) that produced the most rapid increase in p56lck kinase activity also induced the most rapid delocalization of the kinase. In stimulated cells, both p56lck and CD2 receptors are detected in cytoplasmic vesicles. The internalization of p56lck in endocytic vesicles was established by confocal microscopy. By double staining it was shown that only part of the p56lck colocalized with the internalized CD2 receptor suggesting distinct sorting processes. Internalization of p56lck appeared to be specific of CD2 stimulation as: 1) in Jurkat cells triggered with an anti-CD3 mAb, p56lck was not internalized whereas CD3 receptors were completely endocytosed; 2) when cells were stimulated via CD4, the kinase and CD4 receptors remained associated with the plasma membrane. In addition, internalization of p56lck upon stimulation of CD2 receptors was not modified in CD2+/CD3-Jurkat cells indicating that CD3 is not involved in this process. The identification of different subcellular localizations of p56lck in resting and stimulated T cells should represent an important step in the definition of its functional activity.

Possible involvement of a lipocortin in the initiation of DNA synthesis by human endothelial cells.

This work focused on three themes. First, evidence was obtained for the presence of proteins of 34, 35, 32, and 69 kDa immunologically related to lipocortins I, II, V, and VI, respectively, in human umbilical vein endothelial (HUVE) cells. The 69-kDa protein (p69), but not proteins related to lipocortins I, II, and V, exhibited an increased phosphorylation after exposure of cells to basic fibroblast growth factor (bFGF) and phorbol ester PMA. Second, treatment of HUVE cell particulate fractions with EGTA and hydrophobic affinity chromatography in combination with conventional techniques provided extracts rich in p69 and purified p69. p69 from control cells and extracts from control, bFGF-treated, and PMA-treated cells were found to possess anti-phospholipase A2 (PLA2) activity of lipocortin. In contrast, a striking reverse effect occurred when extracts were obtained from cells exposed to bFGF plus PMA. Third, the combination of bFGF and PMA induced a stimulated PLA2-catalyzed release of arachidonic acid in HUVE cells. This arachidonate production was shown to be involved in the decision of cells to enter into DNA synthesis. Taken together, the present results suggest that phosphorylation of p69 is causally involved in the control of commitment to growth in HUVE cells by acting as a coupling mechanism between surface stimuli and arachidonate pathways.

Identification and characterization of phospholipase A2 inhibitory proteins in human mononuclear cells.

Calcium-dependent phospholipid binding and phospholipase A2 inhibitory proteins were isolated from human mononuclear cells. Lipocortins I and II were present whereas lipocortin IV (endonexin I) was not. The other proteins were purified to homogeneity and shown to have molecular masses of 35, 36, 32 and 73 kDa. The 36-kDa and 73-kDa proteins are related, the smaller appears to be part of the larger. The 73-kDa protein is related to the 67-kDa calelectrin and to lipocortin VI; the 32-kDa protein is different from endonexin I but related to chromobindin 7 and to lipocortin V. The 35-kDa protein has been identified by tryptic peptide sequencing as lipocortin III. All these proteins inhibit phospholipase A2 activity in vitro and the three smaller ones inhibit the [3H]arachidonic acid release from prelabelled monocytes induced by the calcium ionophore A23187 in a dose-dependent manner.

[Modifications of protein kinase C activity and phosphorylation of lipocortin I in cultures of pig thyroid cells]. / Modifications de l'activité de la protéine kinase C et de la phosphorylation de la lipocortine I dans les cultures de cellules thyroïdiennes porcines.

When cultured in the absence of thyreostimulin (TSH), thyroid cells lose some of their differentiated functions such as iodide transport and its incorporation into thyroglobulin. In the presence of TSH (0.1 mU/ml), these differentiated functions are preserved ("TSH cells"). The addition of tetradecanoyl phorbol 13 acetate (TPA) inhibits some differentiated functions of the cells and provokes important modifications of bio-signalling pathways. The protein kinase C (pKC) activity, unchanged in "control" and "TSH cells", was dramatically modified in TPA treated cells. After translocation, the pKC activity was down-regulated and the phosphorylation of its endogenous substrates (35-38 kDa) disappeared. Among these substrates, we identified the lipocortin I (LC I) (35 kDa), a phospholipase A2 inhibitory protein related to the Ca2+ binding protein family. By monodimensional electrophoresis (PAGE-SDS) and western-blot, we evidenced the presence of LCI in cytosols and particulate extracts. By 2 dimensional electrophoresis (PAGE-SDS and IEF) and western-blot we identified a phosphorylated and unphosphorylated LCI protein. The phosphorylation of LCI by pKC decreased its isoelectric point from 6.9-6.6. The modifications of pKC activity and LCI phosphorylation and the changes in the bio-signalling pathways can partly account for the loss of differentiation observed in control or TPA treated cells.