Phosphorylation of 40‐S Ribosomal Subunits by cAMP‐Dependent, cGMP‐Dependent and Protease‐Activated Protein Kinases

Grande, Robert W. Del; Traugh, Jolinda A.

doi:10.1111/j.1432-1033.1982.tb19785.x

Cited by 89 publications

(20 citation statements)

References 33 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Therefore, much effort has been directed recently toward characterization of the relevant S6 kinase(s). Several properties of S6 kinase activity from cells stimulated by growth-promoting agents indicate that it differs from protein kinases A and C, both of which are able to phosphorylate S6 in vitro (14,26,39,53). These properties include lack of inhibition by the heat-stable inhibitor protein (2,3,9,15,16,32,40,46,48) and the inability of phospholipids to affect activity (2,9,15,33,40,48).…”

Section: Discussionmentioning

confidence: 99%

Antibodies to Xenopus egg S6 kinase II recognize S6 kinase from progesterone- and insulin-stimulated Xenopus oocytes and from proliferating chicken embryo fibroblasts.

Erikson¹,

Stefanović²,

Blenis³

et al. 1987

Mol. Cell. Biol.

View full text Add to dashboard Cite

Ribosomal protein S6 becomes highly phosphorylated during progesterone-or insulin-induced maturation of Xenopus laevis oocytes. We have previously purified an Mr 92,000 protein as one of the major S6 kinases from Xenopus unfertilized eggs. In this paper we confirm by renaturation of activity from a sodium dodecyl sulfate-polyacrylamide gel that this protein is an S6 kinase. This enzyme, termed S6 kinase II (S6 K II), was used for the preparation of polyclonal antiserum. Immunocomplexes formed with the antiserum and purified S6 K II were able to express kinase activity with the same substrate specificity as that of the purified enzyme, including autophosphorylation of S6 K II itself. The antiserum did not react with S6 kinase I, another major S6 kinase present in Xenopus eggs, which is chromatographicaily distinct from S6 K II. The administration of progesterone to oocytes resulted in a 20-to 25-fold increase in S6 kinase activity in extracts of these cells. Immunocomplex kinase assays done on extracts revealed that anti-S6 K II serum reacted with S6 kinase from progesterone-treated oocytes. This antiserum also reacted with the activated S6 kinase from insulin-stimulated oocytes. In addition, anti-S6 K II serum reacted with activated S6 kinase from chicken embryo fibroblasts stimulated with serum or transformed by Rous sarcoma virus. These results indicate that S6 K II or an antigenically related S6 kinase(s) is subject to regulation by mitogenic stimuli in various cell types.The phosphorylation of ribosomal protein S6 on serine residues is a common response to growth-promoting stimuli in diverse cell systems. In quiescent cultured cells, S6 is primarily unphosphorylated, whereas in stimulated cells it rapidly becomes highly phosphorylated, incorporating up to 4 to 5 mol of phosphate per mol of S6. Stimuli that elicit this response include serum, various growth factors and hormones, tumor-promoting phorbol esters, and the products of many oncogenes (1,3,4,13,22,30,35,(49)(50)(51)(52). In many cases, the increase in S6 phosphorylation has been correlated with enhanced protein kinase activity specific for S6 in extracts of stimulated cells (2,3,9,33,36,37,40,41,48).Meiotic maturation of oocytes is another example in which phosphorylation of S6 is correlated with response to hormonal stimuli. Fully grown Xenopus laevis oocytes are physiologically arrested in prophase of the first meiotic division. Oocytes treated with progesterone or insulin progress through meiotic cell division and arrest at metaphase II as unfertilized eggs, a process termed oocyte maturation. Studies in several laboratories have shown that in Xenopus oocytes S6 undergoes maximal phosphorylation upon maturation induced by treatment with progesterone or insulin or by microinjection of maturation-promoting factor (21,24,34,47). In addition, S6 phosphorylation on serine residues is increased in oocytes after microinjection of the oncogene products of Rous sarcoma virus or Abelson murine leukemia virus or of purified insulin receptor kinase (30, 31, ...

show abstract

Section: Discussionmentioning

confidence: 99%

Antibodies to Xenopus egg S6 kinase II recognize S6 kinase from progesterone- and insulin-stimulated Xenopus oocytes and from proliferating chicken embryo fibroblasts.

Erikson¹,

Stefanović²,

Blenis³

et al. 1987

Mol. Cell. Biol.

View full text Add to dashboard Cite

show abstract

“…In both experiments the final enzyme protein concentration was 1 ug/ml. Calculation of the molarity of ribosomal protein S6 was based upon the assumption that (i) there is one molecule of S6 in each 40S particle, (ii) the extinction coefficient of the 40S particle at A260 is 11.4 mg-', and (iii) the 40S particle Mr is 1.3 x 106 (36). by the Ca2+-phospholipid-dependent protein kinase first followed by the catalytic subunit of the cAMP-dependent protein kinase (A) (arrow).…”

mentioning

confidence: 99%

“…Interestingly, the extent of phosphorylation by the cAMP-dependent protein kinase is somewhat greater when S6 is first phosphorylated by the Ca2+-phospholipid-dependent protein kinase. This-may explain in part why incorporation of phosphate into S6 by the-cAMP~dependent protein kinase has been reported to vary between 1 and 2 mol of P per mol of S6 (36)(37)(38)(39)(40). In intact cells, two sites are phosphorylated in response to cAMP (38).…”

mentioning

confidence: 99%

Purified rat brain calcium- and phospholipid-dependent protein kinase phosphorylates ribosomal protein S6.

Peuch¹,

Ballester²,

Rosen³

1983

Proc. Natl. Acad. Sci. U.S.A.

146

View full text Add to dashboard Cite

The Ca2+-phospholipid-regulated protein kinase has been purified to homogeneity from a 100,000 x g supernatant fluid of rat brain homogenate by a procedure that includes DEAEcellulose chromatography and successive filtrations on Ultrogel AcA 34 in EGTA and in phosphatidylserine and Ca2+. A more rapid purification consisting of DEAE-cellulose chromatography, Ultrogel AcA 34 gel filtration chromatography, and DEAE-trisacryl chromatography, all in the presence of EGTA, was also developed. Although the enzyme obtained by the latter procedure is not homogeneous, it exhibits properties similar to those of the pure enzyme and is more stable. In addition, the DEAE--trisacryl step permitted resolution of a contaminating Ca2+-inhibitable protein kinase that can interfere with studies of the Ca2+-phospholipidstimulated enzyme. The homogeneous enzyme, purified about 300-fold, was estimated to have a Mr of 84,000. Its activity was 20-to 30-fold higher in the presence of phospholipid and Ca2" than in the presence of phospholipid and EGTA, EGTA, or Ca2+ alone. The specific activity of the activated kinase was 852 nmol of P incorporated into histone per min/mg at 20TC. The pure enzyme underwent autophosphorylation in a Ca2+-and phospholipid-dependent manner. This reaction was inhibited in the presence of histones without affecting the kinetic properties of the enzyme. Under optimal assay conditions, the homogeneous enzyme was activated 10-20% by either 10 ImM diolein or 100 nM phorbol 12-myristate 13-acetate. Activation of the purified enzyme by diolein or the phorbol ester was far greater (3-to 4-fold) when aggregated instead of freshly sonicated phospholipids were used, suggesting that these compounds affect the interaction of the enzyme with phospholipids and Ca2+. The purified enzyme catalyzed the phosphorylation of the 40S ribosomal subunit protein S6. The Km, for S6 was %1 ,uM and it was estimated that 2 mol of phosphate were incorporated per mol of S6. The observation that protein S6 can be phosphorylated by the purified Ca2+-phospholipid-dependent protein kinase may link recent reports that phorbol ester tumor promoters activate the Ca2+-phospholipid-dependent protein kinase in vitro and stimulate phosphorylation of the ribosomal protein S6 in vivo.A Ca2+-dependent protein kinase activity that requires phospholipids but not calmodulin was originally described by Takai and colleagues (1, 2) and has been recently purified from bovine heart and from rat brain (3,4). This new type of protein kinase has been found in a wide variety of tissues and phyla (5, 6). Although protein substrates have been observed in brain (7), heart (8-10), pancreas, liver, vas deferens, adrenal (11), and platelets (12, 13), there is thus far no direct evidence that these phosphorylations are involved in cell regulation and the protein substrates themselves have not been identified. Phenothiazines, anesthetics such as dibucaine, and other phospholipidinteracting drugs inhibit the partially purified Ca2+-phospholipid dependent protein kinas...

show abstract

“…7). This behaviour is quite similar to that of the cyclic-AMP-dependent protein kinase [24], but different from that of protein kinase C [2]. It perhaps seems a little strange that two enzymes presumed to be involved in the phosphorylation of ribosomes in vivo should have less than maximal activity at physiological K' concentration.…”

mentioning

confidence: 48%

Induction, partial purification and characterization of a hamster fibroblast protein kinase activity that phosphorylates ribosomal protein S6

Jakubowicz

Leader

1987

Eur J Biochem

View full text Add to dashboard Cite

When BHK cells were grown to confluence and the growth medium replenished, there was a large and rapid increase in the phosphorylation of ribosomal protein S6. In postribosomal extracts of these cells, prepared in the presence of glycerol 2-phosphate and EGTA, a ribosomal protein S6 kinase was detected. The increase in activity of this protein kinase broadly reflected the increase in phosphorylation of ribosomal protein S6 observed in vivo. This ribosomal protein S6 kinase activity was substantially purified by a combination of phosphocellulose, DEAEcellulose, Mono Q and heparin-Sepharose chromatography, and some of its characteristics were examined. When the products of phosphorylation of 40s ribosomal subunits by purified enzyme in vitro were analysed using twodimensional gel electrophoresis, monophosphorylated and diphosphorylated forms of ribosomal protein S6 were observed to be the predominant radioactively labelled species.Ribosomal protein S6 is one of only two phosphoproteins in the ribosomes of eukaryotic cells cultivated under nonpathological conditions, and can acquire up to five seryl phosphoryl groups per molecule [l]. Although there are many situations in which there is a suggestive correlation between the extent of phosphorylation of ribosomal protein S6 and the rate of protein synthesis in vivo, there are also a significant number in which no such correlation obtains (discussed in [2]), and as yet there has been no convincing demonstration of an effect of the phosphorylation on the function of ribosomes in vitro.Nevertheless there is currently much interest in the mechanism(s) by which the phosphorylation of ribosomal protein S6 is increased as a rapid response to growth stimuli such as insulin [3], epidermal growth factor [4] and plateletderived growth factor [5], the initial effects of which are thought to involve activation of the tyrosyl protein kinase activities of their receptors. As a first step in defining this mechanism, attention has focused on identifying and characterizing the ribosomal protein S6 kinase, the activation of which is responsible for the increased phosphorylation. The cyclic-AMP-dependent protein kinase, although probably involved in the phosphorylation of ribosomal protein S6 in other circumstances [6], is not implicated in the action of growth factors. The effect of growth factors on phospholipid turnover [7] suggested that protein kinase C might be a more serious candidate for the ribosomal protein S6 kinase, and it Correspondence to D. P. Leader, Department of Biochemistry, Abbreviations. BHK cells, baby hamster kidney fibroblasts. Definition. AZ6,, unit, the quantity of material in 1 ml solution which has an absorbance of 1 at 260 nm when measured in a cell with a 1 -cm light path.University of Glasgow, Glasgow, Scotland GI2 SQQ Enzyme. Protein kinase (EC 2.7.2.37).has been shown that this enzyme can catalyse the phosphorylation of ribosomal protein S6 in vitro [8, 2, 91. Nevertheless there is no evidence that protein kinase C is involved in the action of insulin [l...

show abstract

Phosphorylation of 40‐S Ribosomal Subunits by cAMP‐Dependent, cGMP‐Dependent and Protease‐Activated Protein Kinases

Cited by 89 publications

References 33 publications

Antibodies to Xenopus egg S6 kinase II recognize S6 kinase from progesterone- and insulin-stimulated Xenopus oocytes and from proliferating chicken embryo fibroblasts.

Antibodies to Xenopus egg S6 kinase II recognize S6 kinase from progesterone- and insulin-stimulated Xenopus oocytes and from proliferating chicken embryo fibroblasts.

Purified rat brain calcium- and phospholipid-dependent protein kinase phosphorylates ribosomal protein S6.

Induction, partial purification and characterization of a hamster fibroblast protein kinase activity that phosphorylates ribosomal protein S6

Contact Info

Product

Resources

About