Subcellular location of stimulus-affected endogenous phosphoproteins in the rat parotid gland

Rat parotid minces were labeled with [32P]Pi, stimulated with isoproterenol, homogenized in sucrose, and fractionated on continuous sucrose density gradients. We analyzed the resulting fractions by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiograms were made from the gels. Comparison of fractions from control and isoproterenol-stimulated minces revealed seven phosphoproteins that were affected by isoproterenol. The subcellular location of these proteins was determined by comparing their distribution in the sucrose gradients with that of a number of enzymes that are characteristic of specific organelles. Isoproterenol decreased the phosphorylation of two cytoplasmic proteins (Mr 16,000 and 18,000) and increased the phosphorylation of a third (Mr 14,000). The phosphorylation of two endoplasmic reticulum proteins was increased by isoproterenol (Mr 20,500 and 22,500), as was an Mr 31,000 protein which was probably the S6 ribosomal protein. The phosphorylation of a secretory granule protein (Mr 24,000) was decreased by isoproterenol. We then developed a purification scheme for parotid secretory granules. By using this method, we demonstrated that the phosphorylation of the Mr 24,000 was also decreased by carbamylcholine. Granules purified by this method also contained a small number of other phosphoproteins whose phosphorylation was increased only by isoproterenol. Secretory granule-associated stimulus-affected phosphoproteins were found in the particulate fraction when the granules were hypotonically lysed, and were not extracted from the particulate fraction by washing with 0.6 M KCl.     

The primary structure of rat ribosomal protein S6

The amino acid sequence of rat ribosomal protein S6, the major phosphoprotein in the organelle, was deduced from the sequence of nucleotides in two recombinant cDNAs and confirmed from the sequence of amino acids in portions of the protein. Ribosomal protein S6 contains 249 amino acids and has a molecular weight of 28,683. The protein has 15 seryl residues; 7 are located in the carboxyl-terminal sequence of 15 amino acids and probably include most if not all of the residues that are phosphorylated. There are related repeated sequences of 10 amino acids each that occur at four separate positions in S6 and that are very basic. Rat S6 is homologous to Saccharomyces cerevisiae S10 (the extent of the identity is 75%) and most likely also to Schizosaccharomyces pombe S6.     

Purification and characterization of multiple S6 phosphatases from the rat parotid gland

S6 phosphatase activities, which dephosphorylate the phosphorylated S6 synthetic peptide, RRLSSLRASTSKSESSQK, were purified to near homogeneity from the membrane and cytosolic fractions of the rat parotid gland. Multiple S6 phosphatases were fractionated on Mono Q and gel filtration columns. In the cytosolic fraction, at least three forms of S6 phosphatase, termed peaks I, II, and III, were differentially resolved. The three forms had different sizes and protein compositions. The peak I enzyme, which had an approximately Mr of 68 kDa on gel filtration, appears to represent a dimeric form of the 39 kDa protein. This S6 phosphatase showed the high activity in the presence of EGTA and was completely inhibited by nanomolar concentrations of either okadaic acid or inhibitor 2. The peak II S6 phosphatase enzyme, with an Mr of 35 kDa, was activated by Mn²⁺. This form could be a proteolytic product of the catalytic subunit of type 1 phosphatase, due to its sensitivities to okadaic acid and inhibitor 2. The peak III enzyme, with an Mr of 55 kDa, is a Mn²⁺-dependent S6 phosphatase. This S6 phosphatase can be classified as a type 1 phosphatase, due to its sensitivity to okadaic acid, since the IC₅₀ of okadaic acid is 4 nM. However, the molecular mass of this S6 phosphatase differs from that of the type 1 catalytic subunit (37 kDa) and showed less sensitivity to inhibitor 2. On the other hand, the membrane fraction contained one form of the S6 phosphatases, termed peak V (Mr 34 and 28 kDa), which could be classified as a type 1 phosphatase. This S6 phosphatase activity was greatly stimulated by Mn²⁺.Abbreviations PP1-C catalytic subunit of type 1 protein phosphatase - SDS sodium dodecyl sulfate - Hepes 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid - PMSF phenylmethylsulfonyl fluoride - Mops 4-morpholine propanesulfonic acid - EDTA ethylenediaminetetraacetate - EGTA [ethylenbis (oxyethylenenitrilo)]-tetra acetic acid     

Binding of rat ribosomal proteins to yeast 5.8S ribosomal ribonucleic acid.

5.8 S RNA-protein complexes were prepared using purified yeast 5.8 S RNA and proteins from the large ribosomal subunit of rat liver. Formation of such hybrid complexes, as measured by Millipore filtration, was dependent on protein concentration. Binding of proteins to the RNA could approach saturation. Such complexes were isolated from sucrose density gradient centrifugation and shown to contain proteins L6, L8, L19, L35 and L35a. These proteins were identified by their molecular weights on polyacrylamide gels containing dodecylsulfate and their mobilities on two dimensional polyacrylamide gels.     

High-performance affinity chromatography for the purification of heparin-binding proteins from detergent-solubilized smooth muscle cell membranes

Heparin and heparan sulfates are regulators of cellular events including adhesion, proliferation and migration. In particular, the antiproliferative effect of heparin on smooth muscle cell (SMC) growth is well described. However, its mechanism of action remains unclear. Numerous results suggest an endocytosis mediated by a still unknown heparin receptor on vascular SMCs. In order to identify a putative heparin receptor on SMCs that could be involved in heparin signalling, affinity chromatography supports were developed. In this paper, we describe high-performance liquid affinity chromatography (HPLAC) supports obtained from silica beads coated with dextran polymer substituted by a calculated amount of diethylaminoethyl functions. With a polysaccharide dextran layer, this type of support can be grafted with specific ligands, such as heparin, using conventional coupling methods. In a previous work, we demonstrated, using butanedioldiglycidyl ether, that silica stationary phases coupled to heparin could be used for the fast elution and good peak resolution of heparin-binding proteins. In the present work, an affinity chromatographic fraction of SMC membrane extracts was analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and six heparin-binding proteins from dodecyloctaethyleneglycol monoether-solubilized SMCs were observed. Their M_r values were between 40 and 70 kDa, with three major protein bands at 66, 45 and 41 kDa. These results indicate the usefulness of the chromatographic method for purifying heparin binding proteins from SMC membrane.     

Ca2+/calmodulin-dependent protein phosphatase in bovine parotid gland: purification and characterization

Calmodulin-dependent protein phosphatase (CaM-PPase) was isolated from bovine parotid gland by sequential application of DEAE-52, Affi-gel blue and calmodulin-affinity chromatography followed by gel filtration and high performance liquid chromatography. The enzyme was activated in the simultaneous presence of Ni2+ or Mn2+ and Ca2+ plus calmodulin. Ca2+/calmodulin-dependent activation of CaM-PPase was antagonized by inhibitors of calmodulin action, such as W-7 and trifluoperazine. Tryptophan fluorescence was quenched in the presence of Ni2+. CaM-PPase was a heterodimer. The molecular weights of large subunits which bound calmodulin (CaM) were 68 kD and 58 kD - the 68 kD subunit was predominant. Polyclonal antibodies against bovine calcineurin cross-reacted with both types of larger subunits. Using polyclonal antibodies against bovine calcineurin or the monoclonal antibody against subunit B of bovine calcineurin, the smaller molecular weight subunit (19 kD) was found to be immunologically identical to subunit B of bovine calcineurin. In bovine parotid gland, CaM-PPase was found both in acinar and duct cells.     

Calcium-dependent inhibition of protein synthesis in rat parotid gland.

1. Protein synthesis in the rat parotid gland in vitro was studied by measuring the incorporation of [3H]phenylalanine into trichloroacetic acid-insoluble proteins. In the unstimulated gland, the rate of incorporation was dependent on the phenylalanine concentration in the medium and proceeded linearly for up to 3h. 2. Adrenaline, carbamoylcholine, phenylephrine and ionophore A23187 inhibited the incorporation of [3H]phenylalanine into acid-insoluble protein; isoprenaline, dibutyryl cyclic AMP and 8-bromo-cyclic GMP were inactive. 3. Inhibition by adrenaline and carbamoylcholine but not by ionophore A23187 required extracellular Ca2+. 4. Both adrenaline and carbamoylcholine increased the magnitude of the acid-soluble [3H]phenylalanine pool at 10 micrometer extracellular phenylalanine, but had no effect if the phenylalanine concentration was increased to 200 micrometer. 5. There was no correlation between cellular ATP content and the observed inhibition of protein synthesis. 6. Our results suggest that both alpha-adrenergic and cholinergic receptors may play a role in the regulation of protein synthesis in the rat parotid gland, and that their effects are mediated by a rise in intracellular free Ca2+.     

Oxidants induce phosphorylation of ribosomal protein S6

We have investigated the phosphorylation of the ribosomal S6 protein which may be on the pathway of mitogenic stimulation in response to oxidants. Mouse epidermal cells JB6 (clone 41) were exposed to active oxygen generated extracellularly by glucose/glucose oxidase (producing H2O2) or xanthine oxidase (producing H2O2 plus superoxide) or active oxygen produced intracellularly by the metabolism of menadione (producing mostly superoxide). All three sources of active oxygen induced rapidly a protein kinase activity which phosphorylated S6 in cellular extracts prepared in the presence of the phosphatase inhibitor beta-glycerophosphate. Maximal activity was reached within 15 min of exposure, and phosphorylation occurred specifically at serine residues. Strong activation of the protein kinase activity was also observed by diamide which selectively oxidizes SH functions. The following observations characterize the reaction: 1) Extracellular addition of catalase but not Cu,Zn-superoxide dismutase was inhibitory, implicating H2O2 rather than superoxide as the active species. 2) Exposure of JB6 cells to reagent H2O2 or H2O2 released by glucose/glucose oxidase resulted in a measurable increase in intracellular free Ca2+. 3) The intracellular Ca2+ complexer quin 2 suppressed the reaction. 4) The calmodulin antagonist trifluoperazine prevented the activation of the protein kinase. 5) Exposure of cells to Mn2+ and La3+, which stimulate calmodulin-dependent activities, potently increased the S6 kinase activity of the cell extracts. 6) Desalted extracts strictly required the addition of Mg2+ and their activity was inhibited by Mn2+. In contrast, the phosphorylation of a 95-kDa protein was strongly stimulated by Mn2+. 7) For several agonists, i.e. active oxygen, phorbol 12-myristate 13-acetate, and serum, tryptic peptide analysis yielded the same phosphopeptides, suggesting that a common S6 kinase is involved in these reactions. From these data we propose that oxidants induce an increase in intracellular free Ca2+ which activates a Ca2+/calmodulin-dependent protein kinase and, as a consequence, an S6 kinase.     

Primary structure of mammalian ribosomal protein S6

Ribosomal protein S6 was isolated from rat liver ribosomes by reversed-phase high-performance liquid chromatography (HPLC) and subjected to cyanogen bromide and proteolytic cleavages. The cleavage fragments were resolved by HPLC and sequenced by automated Edman degradation. The overall amino acid sequence of S6 (249 residues) was determined by alignment of the overlapping sequences of selected cyanogen bromide, chymotryptic, tryptic, and clostripain cleavage fragments. The only protein found to exhibit close homology with the S6 sequence is yeast ribosomal protein S10 (61% sequence identity). Previously, characterized phosphopeptide derivatives of S6 containing phosphorylation sites for adenosine 3',5'-cyclic phosphate dependent and protease-activated protein kinases originate from the carboxy-terminal region of S6 encompassing residues 233-249.     

Identification by affinity chromatography of the eukaryotic ribosomal proteins that bind to 5.8 S ribosomal ribonucleic acid.

The proteins that bind to rat liver 5.8 S ribosomal ribonucleic acid were identified by affinity chromatography. The nucleic acid was oxidized with periodate and coupled by its 3'-terminus to Sepharose 4B through and adipic acid dihydrazide spacer. The ribosomal proteins that associate with the immobilized 5.8 S rRNA were identified by polyacrylamide gel electrophoresiss: they were L19, L8, and L6 from the 60 S subunit; and S13 and S9 from the small subparticle. Small amounts of L14, L17', L18, L27/L27', and L35', and of S11, S15, S23/S24, and S26 also were bound to the affinity column, but whether they associate directly and specifically with 5.8 S rRNA is not known. Escherichia coli ribosomal proteins did not bind to the rat liver 5.8 S rRNA affinity column.     

Estrogen-binding protein from rat preputial gland: purification and characterization.

Cytosol from the rat preputial gland has been shown to contain a protein which binds both estrone and estradiol. The protein, after a 26-fold purification from the cytosol of female Sprague-Dawley rats, migrated as one band during electrophoresis in sodium dodecyl sulfate on acrylamide gel. The electrophoretic mobility indicated a molecular weight of 15,000. The association constant for estrone as determined by equilibrium dialysis was 1.2 X 10(7) M-1, while that for 17beta-estradiol was 3.3 X 10(6) M-1. Progesterone, cortisol, testosterone, or diethylstilbestrol did not bind to the purified protein, whereas 17alpha-estradiol or estriol bound only slightly. In the presence of retinoic acid, but not retinol, the binding of estrone was reduced. Optimum binding for estrone was at pH 6.5 to 8.5.     

Subcellular distribution of ribosomal proteins S6 and eL12

Summary The process of ribosome assembly in eukaryotes was studied by injecting tritium-labeled ribosomal proteins S6 and eL12 into oocytes of Xenopus laevis. The subcellular distribution of the two proteins was visualized by means of autoradiography in sections of oocytes. Protein S6 but not eL12 was found in the nucleus where it accumulated at the nucleoli. In the presence of actinomycin D the accumulation of S6 at the nucleoli was reduced. In-situ immunofluorescence studies indicated that S6 is located at the nucleoli and eL12 exclusively in the cytoplasm. It appears that S6 is involved in the early ribosomal assembly process at the nucleoli, whereas eL12 is restricted to the cytoplasm where it is incorporated into 60S ribosomal subunits in a late assembly step.     

Rapid separation of Escherichia coli 30S ribosomal proteins by fast protein liquid chromatography (FPLC)

The proteins of the 30S ribosomal subunit from Escherichia coli have been separated by reverse-phase high-performance liquid chromatography on a short alkyl chain (C1/C8)-coated phase. The reverse-phase column was connected to a fast protein liquid chromatography (FPLC) system. The 21 proteins of the 30S ribosomal subunit were resolved into 16 peaks. Eleven proteins were isolated in purified form in a single chromatographic run as shown by polyacrylamide gel electrophoresis and amino acid analysis. Interestingly, the retention times of some proteins differed from the retention times observed on other reversed-phase support materials. The results show the speed and resolution of reverse-phase FPLC for both analytical and semi-preparative separations of 30S ribosomal proteins.     

cAMP-dependent protein phosphorylation of membrane proteins in the parotid gland, platelets and liver. Comparison of a 22-kDa phosphoprotein from rat parotid microsomes (protein III) with phosphoproteins of similar molecular size from platelet and liver membranes

Stimulation of secretion in exocrine secretory glands leads to the phosphorylation of a 22-kDa membrane protein (protein III) whose function is still unknown [Jahn et al. (1980) Eur. J. Biochem. 112, 345-352; Jahn & S?ling (1980) Proc. Natl Acad. Sci. USA 78, 6903-6906]. This report describes the comparison of this protein with phosphorylated membrane proteins of similar molecular mass in platelets and liver. Incubation of platelets with agents which raise the intracellular cAMP concentration results in the phosphorylation of a 22-kDa protein which is also phosphorylated in membrane preparations by endogenous kinases or by exogenous cAMP-dependent protein kinase. It is shown that this protein is distinct from protein III although both proteins have the same molecular mass and are substrates of cAMP-dependent protein kinase. In contrast to platelets, protein III could be demonstrated in liver microsomes. This indicates that the function of protein III is not exclusively linked to the stimulus-secretion coupling in exocrine cells.     

Interferon-gamma engages the p70 S6 kinase to regulate phosphorylation of the 40S S6 ribosomal protein

The signals generated by the IFNgamma receptor to initiate mRNA translation and generation of protein products that mediate IFNgamma responses are largely unknown. In the present study, we provide evidence for the existence of an IFNgamma-dependent signaling cascade activated downstream of the phosphatidylinositol (PI) 3'-kinase, involving the mammalian target of rapamycin (mTOR) and the p70 S6 kinase. Our data demonstrate that p70 S6K is rapidly phosphorylated and activated during engagement of the IFNgamma receptor in sensitive cell lines. Such activation of p70 S6 kinase is blocked by pharmacological inhibitors of the PI 3' kinase and mTOR, and is abrogated in double-knockout mouse embryonic fibroblasts for the alpha and beta isoforms of the p85 regulatory subunit of the PI 3'-kinase. The IFNgamma-activated p70 S6 kinase subsequently phosphorylates the 40S S6 ribosomal protein on serines 235/236, to regulate IFNgamma-dependent mRNA translation. In addition to phosphorylation of 40S ribosomal protein, IFNgamma also induces phosphorylation of the 4E-BP1 repressor of mRNA translation on threonines 37/46, threonine 70, and serine 65, sites whose phosphorylation is required for the inactivation of 4E-BP1 and its dissociation from the eukaryotic initiation factor-4E (eIF4E) complex. Thus, engagement of the PI 3'-kinase and mTOR by the IFNgamma receptor results in the generation of two distinct signals that play roles in the initiation of mRNA translation, suggesting an important role for this pathway in IFNgamma signaling.