Raymond Lecocq

Tumor necrosis factor-α induces the phosphorylation of 28kDa stress proteins in endothelial cells: Possible role in protection against cytotoxicity?

Tumor necrosis factor-alpha has been shown to rapidly increase the phosphorylation of three 28 kDa proteins in bovine aortic endothelial cells but not in L929 cells. Tumor necrosis factor-alpha induces the necrosis of the latter cells but not of the former. Arsenite enhanced the phosphorylation of the same 28kDa proteins as tumor necrosis factor-alpha in the endothelial cells. As stress proteins often play a protective role, we suggest that the phosphorylation of these proteins in endothelial cells may be responsible for the resistance of these cells to tumor necrosis factor-alpha.

Cloning and sequencing of a calcium-binding protein regulated by cyclic AMP in the thyroid.

p24 is a thyroid protein (Mr 24,000) identified by two‐dimensional gel electrophoresis on the basis that its synthesis and phosphorylation are up‐regulated by thyrotropin and cyclic AMP agonists. p24 cDNA was cloned from a lambda gt11 cDNA library using a polyclonal antibody raised against the protein recovered from a Western blot spot. The encoded polypeptide (189 residues) displays a putative target‐site for phosphorylation by cyclic AMP‐dependent protein kinase and belongs to the superfamily of proteins binding Ca2+ through ‘EF hand’ domains. It presents four such domains of which two agree closely with the consensus. The ability of p24 to bind Ca2+ has been directly confirmed on Western blots. p24 was detected in many tissues including the salivary glands, the lung and the brain. The ubiquitous nature of p24, together with its regulatory and sequence characteristics suggest that it constitutes an important target common to the cyclic AMP and Ca2+‐phosphatidylinositol cascades.

Arginine 343 and 350 Are Two Active Site Residues Involved in Substrate Binding by Human Type I D-myo-Inositol 1,4,5-Trisphosphate 5-Phosphatase

The crucial role of two reactive arginyl residues within the substrate binding domain of human Type I D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P) 5-phosphatase has been investigated by chemical modification and site-directed mutagenesis. Chemical modification of the enzyme by phenylglyoxal is accompanied by irreversible inhibition of enzymic activity. Our studies demonstrate that phenylglyoxal forms an enzyme-inhibitor complex and that the modification reaction is prevented in the presence of either Ins(1,4,5)P, D-myo-inositol 1,3,4,5-tetrakisphosphate (Ins(1, 3,4,5)P) or 2,3-bisphosphoglycerate (2,3-BPG). Direct [3H]Ins(1,4,5)P binding to the covalently modified enzyme is dramatically reduced. The stoichiometry of labeling with 14C-labeled phenylglyoxal is shown to be 2.1 mol of phenylglyoxal incorporated per mol of enzyme. A single [14C]phenylglyoxal-modified peptide is isolated following α-chymotrypsin proteolysis of the radiolabeled Ins(1,4,5)P 5-phosphatase and reverse-phase high performance liquid chromatography (HPLC). The peptide sequence (i.e. M-N-T-R-C-P-A-W-C-D-R-I-L) corresponds to amino acids 340-352 of Ins(1,4,5)P 5-phosphatase. An estimate of the radioactivity of the different phenylthiohydantoin amino acid derivatives shows the modified amino acids to be Arg-343 and Arg-350. Furthermore, two mutant enzymes were obtained by site-directed mutagenesis of the two arginyl residues to alanine, and both mutant enzymes have identical UV circular dichroism (CD) spectra. The two mutants (i.e. R343A and R350A) show increased K values for Ins(1,4,5)P (10- and 15-fold, respectively) resulting in a dramatic loss in enzymic activity. In conclusion, we have directly identified two reactive arginyl residues as part of the active site of Ins(1,4,5)P 5-phosphatase. These results point out the crucial role for substrate recognition of a 10 amino acids-long sequence segment which is conserved among the primary structure of inositol and phosphatidylinositol polyphosphate 5-phosphatases.

The use of L-[35S]methionine and L-[75Se]selenomethionine for double-label autoradiography of complex protein patterns on two-dimensional polyacrylamide gels: a drastic shortening of the exposure time.

Abstract A method of autoradiography that allows discrimination between 35S- and 75Se-labeled material on polyacrylamide gels is described. It involves the incorporation of l -[35S]methionine and l -[75Se]selenomethionine into proteins. The autoradiographic exposure times are decreased at least sixfold with regard to the previously described techniques using 3H- and 14C-labeled proteins.

Cloning and sequence analysis of human calcyphosine complementary DNA

Calcyphosine, initially identified as thyroid protein p24, is a calcium-binding protein containing four EF-hand domains. It was first cloned and characterized in the dog and corresponds to R2D5 antigen in rabbit. Using the canine calcyphosine cDNA sequence as a probe, we have isolated its human counterpart from a thyroid cDNA library. The two sequences display a high degree of conservation, both at nucleotide and deduced amino acid levels. Sequence comparison with other proteins showed that the closest homologue of calcyphosine is the crustacean CCBP-23 protein. Northern blot analysis revealed that calcyphosine messenger RNA is much less abundant in human than in canine thyrocytes. Western blot experiments indicated that the amount of protein is also dramatically reduced in man compared to dog.

In vivo and in vitro effects of thyrotropin on ribosomal pattern of dog thyroid

Abstract The proportion of ribosomes bound to mRNA ( i.e. polysomes) was expressed as the P/P+M ratio, where P and M were the polysomes and monosomes area, respectively, measured on a ribosomal density gradient centrifugation pattern. Thyrotropin both in vivo and in vitro enhanced this P/P+M ratio. The average increase of P/P+M by thyrotropin was 20 % ( P ). Thyrotropin also increased the H/H+L ratio, where H represents the heavy polysome population ( > 6 ) and L the light polysome population (14 %; P ). These effects are specific for thyrotropin, they are observed within 30 min of adding thyrotropin and they are obtained for concentrations of 1 munit/ml or higher. Cyclic AMP and dibutyryl cyclic AMP reproduced the stimulation. There is a close parallelism between these effects of thyrotropin and the activation of protein synthesis by this hormone. The rapid response to thyrotropin, as well as labeling experiments with [ 3 H]uridine, support the hypothesis that these effects of thyrotropin bear on translation and not on transcription. It is suggested that thyrotropin rapidly activates protein synthesis in dog thyroid slices by a process mediated by cyclic AMP which bears on translation either by unmasking of heavy mRNA or by increasing the rate of initiation of protein synthesis.

Translation in Xenopus oocyte of messenger RNA coding for a beef heavy-chain thyroglobulin.

Abstract Poly(A)-rich RNA extracted from membrane-bound thyroid polysomes has been shown to contain two fractions showing thyroglobulin messenger activity: a heavy 33 S fraction and a 16–18 S fraction. When injected into Xenopus oocyte, the 33 S mRNA specifically promoted the synthesis of a polypeptide chain of about 185 000 daltons as revealed by electrophoresis in sodium dodecylsulphate—polyacrylamide gel after reduction and alkylation. Our results strongly support models of thyroglobulin structure involving the existence of heavy thyroglobulin subunits.

Calcium binding protein calcyphosine in dog central astrocytes and ependymal cells and in peripheral neurons.

Calcyphosine is a calcium binding protein discovered in the dog thyroid in 1979. Calcyphosine mRNA and immunoreactivity were detected using Western and Northern blotting in the cerebral cortex, cerebral white matter and cerebellum. Using immunohistochemistry and in situ hybridization, both are present in ependymal cells, choroid plexus cells and several types of astrocytes of the subependymal cerebral layer, the cerebellar Bergmann layer, the retinal ganglion cell layer, the optic nerve and the posterior pituitary. Both are also present in neurons of nasal olfactory mucosa, enteric Auerbach and Meissner plexuses, orthosympathic and spinal cord ganglia as well as in endocrine cells of neural crest origin in the adrenal medulla. Calcyphosine immunoreactive astrocytes were also present mainly in hemispheric cerebral gray and white matter, hemispheric subcortical structures, brain stem and spinal cord. These results show that calcyphosine is a characteristic calcium binding protein of astrocytes and ependymal cells in the central nervous system and of neurons in the peripheral nervous system. This is of interest in view of the importance of calcium regulation in these cells, and since calcyphosine a calcium binding protein phosphorylated by cAMP dependent process, may be an intermediate between cAMP and inositol phosphate cascades.

Carbamylcholine and thyrotropin activate distinctive pathways of protein phosphorylation in dog thyroid

The role of protein phosphorylation in the regulation of thyroid function by carbamylcholine was investigated using dog thyroid slices incubated in the presence of [32P]phosphate and two-dimensional electrophoresis. In these intact cells, carbachol increased the phosphorylation of three polypeptides with Mr values of 21500, 24 000 and 29000. Maximal [32P]phosphate incorporation occurred within 5 min of addition of carbamylcholine and was still observed after 10 min of action of this agent. Incubation of dog thyroid slices with thyrotropin for 10 min increased the phosphorylation of 11 polypeptides which were identical to those observed previously after 2 h of hormone action (Lecocq, R., Lamy, F. and Dumont, J.E. (1979) Eur. J. Biochem. 102, 147-152). All three polypeptides whose phosphorylation is increased by carbamylcholine were different from those whose phosphorylation is increased by thyrotropin. Under our experimental conditions, the calcium ionophore A23187 did not stimulate significantly [32P]phosphate incorporation in these three polypeptides. In conclusion, our results show that carbamylcholine and thyrotropin, which have some antagonist and some similar effects on dog thyroid, do not act through the phosphorylation of the same proteins. Although we have, in our previous paper, established that a rise in intracellular cyclic AMP could account for the effect of thyrotropin on protein phosphorylation, the nature of the intracellular mediator of carbamylcholine action on this parameter is still uncertain.

Production of dog calcyphosine in bacteria and lack of phosphorylation by the catalytic subunit of protein kinase A in vitro

Calcyphosine is a calcium-binding protein containing four EF-hand domains that is found in several epithelia and in some cells of the central nervous system. In thyroid follicular cells, calcyphosine is synthesized and phosphorylated in response to stimulation by thyrotropin and cAMP agonists. The cDNA coding for dog calcyphosine has been expressed in bacteria under the control of the T7 promoter. Recombinant calcyphosine was purified from crude bacterial lysates by a combination of anion-exchange and hydrophobic interaction chromatography. Phosphorylation assays using the purified catalytic subunit of protein kinase A and the recombinant or the native calcyphosine revealed that, contrary to a previous report, calcyphosine is not significantly phosphorylated by this enzyme in vitro.