Jean-Paul Capony

A Novel Phosphorylation-Dependent RNase Activity of GAP-SH3 Binding Protein: a Potential Link between Signal Transduction and RNA Stability

ABSTRACT A potential p120 GTPase-activating protein (RasGAP) effector, G3BP (RasGAP Src homology 3 [SH3] binding protein), was previously identified based on its ability to bind the SH3 domain of RasGAP. Here we show that G3BP colocalizes and physically interacts with RasGAP at the plasma membrane of serum-stimulated but not quiescent Chinese hamster lung fibroblasts. In quiescent cells, G3BP was hyperphosphorylated on serine residues, and this modification was essential for its activity. Indeed, G3BP harbors a phosphorylation-dependent RNase activity which specifically cleaves the 3′-untranslated region of human c-myc mRNA. The endoribonuclease activity of G3BP can initiate mRNA degradation and therefore represents a link between a RasGAP-mediated signaling pathway and RNA turnover.

The cyclic AMP-dependent modulation of cardiac sarcolemmal slow calcium channels.

Abstract The epinephrine-induced abbreviation of systole is well explained by the cAMP-dependent phosphorylation of phospholamban, the activator of the cardiac sarcoplasmic reticulum calcium pump, and of the inhibitory component of the troponin complex. In contrast, the molecular mechanism of the β-agonist inotropic effects remained unclear until the recent finding that the depolarization-induced Ca 2+ uptake by cardiac sarcolemmal vesicles, the in vitro equivalent of the slow Ca 2+ channel, is activated upon cAMP-dependent phosphorylation of a sarcolemmal integral protein, calciductin [ 34 ]. This paper provides additional evidence for this mechanism by showing a linear correlation between calciductin phosphorylation and voltage-dependent Ca 2+ uptake. At no detectable radiolabelling with ( 32 P)-phosphate, the latter is still ca 28% of its maximal value, suggesting the possibility of residual unlabeled protein-bound phosphate. Calciductin is an excellent substrate of cAMP-dependent protein kinase, since low levels of its catalytic subunit ( ca 6% of the subunit liberated after complete dissociation of the enzyme) are capable of submaximally phosphorylating calciductin and activating Ca 2+ uptake. The Ca 2+ channels of phosphorylated vesicles are similar to those of non-phosphorylated vesicles and of other excitable cells in their inhibition by the calcium antagonist verapamil, lanthanum and other divalent cations. In contrast to the voltage-dependent Ca 2+ uptake, Na + Ca 2+ exchange was not modulated by cAMP-dependent phosphorylation. Sarcolemmal proteolipids were entirely extracted by acidic organic solvent mixtures and purified by high performance liquid chromatography. They made up 2% (w/w) of sarcolemmal proteins and contained essentially calciductin (97%) together with a 14 000 to 16 000 dalton component (3%). Sarcolemmal proteolipids were phosphorylated on seryl residues only. Their amino acid composition indicated the presence of a large number of acidic and hydrophobic residues, accounting for the behaviour of calciductin as an acidic integral proteolipid, similar to phospholamban.

A purified complex from Xenopus oocytes contains a p47 protein, an in vivo substrate of MPF, and a p30 protein respectively homologous to elongation factors EF-1γ and EF-1β

A high molecular mass complex isolated from Xenopus laevis oocytes contains three main proteins, respectively p30, p36 and p47. The p47 protein has been reported to be an in vivo substrate of the cell division control protein kinase p34cdc2. From polypeptide sequencing, we now show that the p30 and the p47 correspond to elongation factor EF‐1β and EF‐1γ. Furthermore, the p30 and p36 proteins were phosphorylated in vitro by casein kinase II.

Changes in the activity of the maturation-promoting factor during meiotic maturation and following activation of amphibian and starfish oocytes: Their correlations with protein phosphorylation

Changes in the extent of protein phosphorylation and their possible correlation with changes in the activity of maturation-promoting (MPF) factor were investigated throughout meiotic maturation and following activation of amphibian and starfish oocytes. Despite several exceptions in the pattern of phosphorylation of individual proteins, high and low levels of protein phosphorylation were found to be correlated with high and low levels of MPF activity. Both the extent of protein phosphorylation and MPF activity were found to drop upon parthenogenetic activation and to cycle synchronously thereafter in the amphibian. In contrast no drop in MPF activity or in the extent of protein phosphorylation was observed following activation of starfish oocytes with ionophore A23187. This suggests that changes of protein phosphorylation and of MPF activity are rather related to the progression of the cell cycle than directly to Ca2+-dependent activation reaction. In amphibians global protein kinase activity in homogenates was found to drop with MPF activity following activation. Changes in the ratio of threonine vs serine phosphorylation were also investigated during the course of meiotic maturation and activation in both amphibian and starfish oocytes: changes in the activity of MPF were found to be better correlated with changes in threonine than serine phosphorylation.

A rapid microdetermination of phosphoserine, phosphothreonine, and phosphotyrosine in proteins by automatic cation exchange on a conventional amino acid analyzer

A cation-exchange chromatographic method for the separation and determination of phosphoserine, phosphothreonine, and phosphotyrosine in proteins after partial acid hydrolysis is described. The short column (0.6 X 8 cm) of an automatic amino acid analyzer was used and elution was carried out isocratically with 10 mM trifluoroacetic acid. The method is highly sensitive and each of the three O-phosphoamino acids can be accurately determined down to the 50-pmol level. Higher sensitivity may be obtained by the use of [32P]phosphate-labeled proteins. A correction factor for the decomposition of phosphoserine or phosphothreonine during acid hydrolysis can be deduced from the amount of inorganic phosphate recovered at the column void volume. The method is sensitive enough to be used for 32P-labeled proteins isolated by two-dimensional gel electrophoresis.

Isolation of cardiac membrane proteolipids by high pressure liquid chromatography. A comparison of reticular and sarcolemmal proteolipids, phospholamban and calciductin.

Membrane-bound phosphorylatable proteolipids were reported to play a role in the regulation of transmembrane Ca2+ fluxes by catecholamines. A generally applicable purification procedure is described by which such proteolipids as the cardiac sarcoplasmic reticulum phospholamban is purified by solvent extraction followed by high pressure liquid chromatography on microparticulate silica. Phospholamban is thereby purified with a yield of 3.37 mg from 100 mg of sarcoplasmic reticulum proteins, significantly higher than that obtained by any of the previously reported procedures. It appeared homogeneous upon dodecyl sulfate-polyacrylamide gel electrophoresis where it is stained by Coomassie blue and detected by autoradiography. The same procedure is applicable to cardiac sarcolemmal calciductin. Both proteolipids exhibit the same Mr 11 000 and pI 3.7 upon two-dimensional gel electrophoresis. Their amino acid compositions are very similar if not identical. This raises the intriguing possibility that phospholamban and calciductin are identical though they obviously belong to different membranes.

Phosphorylation of ribosomal proteins during meiotic maturation and following activation in starfish oocytes: its relationship with changes of intracellular pH.

An increased phosphorylation of ribosomal protein S6 has been shown to be correlated with an increase of intracellular pH (pHi) and with stimulation of protein synthesis in many systems. In this research changes in ribosome phosphorylation following hormone-induced meiotic maturation and fertilization or activation by ionophore A23187 were investigated in starfish oocytes. The hormone was found to stimulate, even in the absence of external Na+, the phosphorylation on serine residues of an Mr 31,000 protein identified as S6, as well as that of an acidic Mr 47,000 protein, presumably S1, on threonine residues. Phosphorylation of ribosomes was an early consequence of hormonal stimulation and did not decrease after completion of meiotic maturation. Fertilization or activation by ionophore of prophase-arrested oocytes also stimulated ribosome phosphorylation. Only S6 was labeled in this case, but to a lesser extent than upon hormone-induced meiotic maturation. Changes in pHi were monitored with ion-specific microelectrodes throughout meiotic maturation and following either fertilization or activation. The pHi did not change before germinal vesicle breakdown (GVBD) following hormone addition, but it increased before first polar body emission. It also increased following fertilization or activation by ionophore or the microinjection of Ca-EGTA. In all cases, alkalinization did not depend on activation of an amiloride-sensitive Na+/H+ exchanger. Microinjection of an alkaline Hepes buffer or external application of ammonia, both of which increased pHi, prevented unfertilized oocytes from arresting after formation of the female pronucleus and induced chromosome cycling. Phosphorylation of S6 was still observed following fertilization or induction of maturation when pHi was decreased by external application of acetate, a treatment which suppressed the emission of polar bodies. Protein synthesis increased in prophase-arrested oocytes after fertilization or activation. It also increased after ammonia addition, although this treatment did not stimulate S6 phosphorylation.

Sequence Homologies between p36, the substrate of pp60src tyrosine kinase and a 67 kDa protein isolated from bovine aorta

A 67 kDa actin-binding protein was isolated from bovine aorta. Partial amino acid sequence determination of two large thermolysin peptides were used to compare 67 kDa bovine aorta protein and p36 the substrate of pp60src tyrosine kinase. Sequence analysis shows that 67 kDa bovine aorta protein shares common domains with p36 and possesses the consensus aminoacid sequences of mammalian Ca2+-dependent membrane-binding protein and p36/gelsolin.

The primary structure of the major parvalbumin from hake muscle. Tryptic peptides derived from the S-sulfo and the performic-acid-oxidized proteins.

With the aim of establishing a starting point for the determination of the complete amino acid sequence of the major parvalbumin from hake muscle, the peptides resulting from the tryptic digestion of the 8-sulfo-derivative and of the performic-acid-oxidized protein were examined. A total of 20 peptides were thus obtained in sufficient quantity and in a state of purity adequate for further studies. Their complete or partial sequence is reported. When due account is taken of obvious overlaps, the total of these peptides fully accounts for the composition of the whole molecule with the exception of one alanine residue, and for the number of peptides expected from the presence of 12 lysines and 1 arginine in this parvalbumin.

High levels of HMG1-2 protein expression in the cytoplasm and nucleus of hydrocortisone sensitive amphibian thymocytes

Summary— A major 26 kDa protein was identified in the cytoplasmic and nuclear compartments of axolotl thymocytes. A polyclonal antiserum was produced against the denatured form of this protein. High levels of 26 kDa were expressed by hydrocortisonesensitive lymphocytes which represent a major thymocyte subpopulation in young animals. However, no further expression of the 26 kDa protein was observed in involuted thymus of adult animals nor in thymus of young artificially metamorphosed axolotls. The 26 kDa was never expressed by splenic and blood peripheral lymphocytes at any stage of development. Partial N‐terminal amino acid sequence and amino acid composition demonstrate that the 26 kDa polypeptide is strongly homologous to HMGI‐2 proteins, the most abundant members of the high mobility group (HMG) non‐histone chromosomal proteins. HMG1‐2 are thought to be involved in the organization of chromatin structure, as well as in the stability, replication and transcription of DNA. It was confirmed that the 26 kDa axolotl polypeptide is recognized by a well characterized rabbit antiserum to rat HMG1‐2 proteins.