Regulation of a ribosomal protein S6 kinase activity by the Rous sarcoma virus transforming protein, serum, or phorbol ester.

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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, ...

Section: Resultsmentioning

confidence: 99%

mentioning

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

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“…These include protease-activated kinase 11 (12,31) and protein kinase C, a Ca2+-activated kinase which may also be phospholipid dependent (11,29), both of which seem to have a fairly broad substrate spectrum. Finally, several laboratories have recently purified a kinase which is apparently specific for S6 and distinct from the kinases described above by a number of criteria (3,10,27,38). This kinase has substantially more activity in extracts from cells in which S6 phosphorylation has been stimulated by insulin (38), epidermal growth factor (EGF) (27), or expression of the oncogene src (3).…”

Section: Resultsmentioning

confidence: 99%

“…Finally, several laboratories have recently purified a kinase which is apparently specific for S6 and distinct from the kinases described above by a number of criteria (3,10,27,38). This kinase has substantially more activity in extracts from cells in which S6 phosphorylation has been stimulated by insulin (38), epidermal growth factor (EGF) (27), or expression of the oncogene src (3).…”

Section: Resultsmentioning

confidence: 99%

Phosphorylation of the Saccharomyces cerevisiae equivalent of ribosomal protein S6 has no detectable effect on growth.

Johnson¹,

Warner²

1987

The phosphorylation of mammalian ribosomal protein S6 is affected by a variety of agents, including growth factors and tumor promoters, as well as by expressed oncogenes. Its potential role in the regulation of protein synthesis has been the object of much study. We have developed strains of Saccharomyces cerevisiae in which the phosphorylatable serines of the equivalent ribosomal protein (S10) were converted to alanines by site-directed mutagenesis. The S10 of such cells is not phosphorylated. Comparison of these cells with the parental cells, whose genomes differ by only six nucleotides, revealed no differences in the lag phase or logarithmic phase of a growth cycle, in growth on different carbon sources, in sporulation, or in sensitivity to heat shock. We conclude that in S. cerevisiae the phosphorylation of ribosomal protein S10 may play no role in regulating the synthesis of proteins. This conclusion leads one to ask whether certain protein phosphorylations are simply the adventitious, if easily observable, result of the imperfect specificity of one or another protein kinase.Ribosomal protein S6 is the major phosphoprotein of eucaryotic ribosomes. It can be phosphorylated at multiple sites (two in Saccharomyces cerevisiae and at least five in vertebrates), and the degree of phosphorylation is somehow related to cell proliferation. A variety of stimuli and agents have been observed to affect the phosphorylation of S6, including insulin (35,40,44), growth factors (40), serum treatment (40), viral transformation (7), phorbol esters (3), and fertilization (2, 26). Phosphorylation of the S6 protein is carried out by a number of kinases, one of which is apparently specific for S6, and is activated by at least two regulatory mechanisms (4).In S. cerevisiae the homologous ribosomal protein, referred to as S10, is phosphorylated after transfer of a stationary culture to fresh nutrient medium (16). Phosphorylation also occurs at an early stage of germination (37). As in other eucaryotes, the protein is dephosphorylated during heat shock (16).At present, much is known about the phenomenology of S6 phosphorylation, but little about its function. Generally, as phosphorylation of protein S6 increases, the level of protein synthesis increases (14, 40), and as dephosphorylation of S6 occurs, the level of protein synthesis decreases (13, 34). However, phosphorylation of S6 has been observed to be stimulated under conditions in which protein synthesis increased, decreased, or remained unchanged (14,17,41). In serum-stimulated 3T3 cells, ribosomes containing highly phosphorylated S6 are preferentially incorporated into new polysomes, suggesting a role in mRNA recruitment (8,40). Such a role is supported by the localization of S6 in the mRNA-binding site of 80S ribosomes (39). In vitro attempts to demonstrate an effect of S6 phosphorylation have been successful in some cases (6) and unsuccessful in others (20,25).Because of the potential importance of the phosphorylation of S6 to the regulation of cell growth and because of th...

“…t Present address: Department of Molecular Biology, Northwestern University Medical College, Chicago, IL 60611. growth is arrested by serum deprivation (48), contact inhibition, or heat shock (21). Phosphorylation of S6 is induced by a wide variety of mitogens and oncogene products, including insulin (31,39,46,47,50), insulinlike growth factors (23), epidermal growth factor (36, 37, 47), plateletderived growth factor (PDGF) (34), fibroblast growth factor (38), tumor-promoting phorbol esters (5,8,50), and transforming gene products of Rous sarcoma virus (5,8,17), Abelson murine leukemia virus (30), simian virus 40, and polyomaviruses (4,28). Increased phosphorylation of S6 is also seen in Xenopus laevis oocytes after progesterone or maturation-promoting factor stimulation of meiotic maturation (33).…”

mentioning

confidence: 99%

Polyomavirus middle T antigen induces ribosomal protein S6 phosphorylation through pp60c-src-dependent and -independent pathways.

Talmage¹,

Blenis²,

Benjamin³

1988

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Phosphorylation of ribosomal protein S6 is elevated in polyomavirus-infected cells. This elevation results only in part from activation of S6 kinase activity. These effects appear to reflect independent activities of wild-type middle T antigen. Hr-t mutant NG59, encoding a defective middle T protein, and mutant Py8O8A, encoding no middle T protein, were unable to induce S6 kinase activity or elevate S6 phosphorylation. Two other site-directed mutants encoding altered middle T proteins did elevate S6 phosphorylation while only weakly stimulating S6 kinase activity. These results suggest at least two independent pathways leading to elevation of S6 phosphorylation. One pathway leads to induction of S6 kinase activity following activation of pp60`cs by transformation-competent middle T antigen. Another pathway operates independently of S6 kinase induction and can be regulated by transformation-defective middle T mutants such as Py1387T. This mutant, encoding a truncated middle T protein that failed to associate with the plasma membrane and to activate pp60C"sc, caused increased levels of S6 phosphorylation without detectably increasing S6 kinase activity. The ability of mutants such as Py1387T to induce S6 phosphorylation correlated with their ability to increase phosphorylation of VP1, an event linked to maturation of infectious virions.Transformation of cells by polyomavirus requires the interaction of the polyomavirus middle T antigen with the cellular proto-oncogene product pp6Oc-src (15,16). This interaction leads to the activation of the intrinsic tyrosine kinase activity of pp60c-src and ultimately to transformation (10,14). Although pp60c-src activation is obligatory for transformation, mutant middle T's exist which interact with and activate pp6Oc-src yet fail to transform infected cells. Full transformation in culture and tumor induction in mice may require the presence of an additional activity in middle T-pp60csrc immune complexes which phosphorylates phosphatidylinositol (PI) (27, 51; D. Talmage, R. Freund, A. Young, C. Dawe, and T. Benjamin, manuscript in preparation).Polyomavirus hr-t mutants encoding defective middle T and small T antigens grow poorly on NIH 3T3 cells, yielding bursts which are only 2 to 5% the size of those of wild-type virus (22). The low burst size results from inefficient or unstable assembly of viral components into infectious virions and is associated with underphosphorylation of the major viral capsid protein, VP1 (19,20). Thus, the role of the hr-t gene in viral growth lies in inducing cellular "permissivity factors" which act, at least in part, to bring about stable phosphorylation of VP1. Activation of this phosphorylation pathway in NIH 3T3 cells does not require detectable interaction of middle T with pp60c-src and therefore represents a middle T function which is separate from its known function during transformation (R. Garcea, D. Talmage, R. Freund, A. Harmatz, and T. Benjamin, manuscript in preparation).The ribosomal protein S6 is highly phosphorylated in growing cell...