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

Abstract

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, ...

A Xenopus ribosomal protein S6 kinase has two apparent kinase domains that are each similar to distinct protein kinases.

“…Studies with this antiserum have revealed that progesterone-or insulin-stimulated oocytes contain an antigenically related enzyme. Furthermore, anti-S6KII antiserum reacts with an S6 kinase activity found in extracts of chicken embryo fibroblasts that had been stimulated with serum or transformed by Rous sarcoma virus (13). These results indicate that S6KII or an antigenically related S6 kinase(s) is subject to mitogenic stimulation in various cell types.…”

“…This event is mediated by a protein kinase activity that can be detected readily by phosphorylation of 40S subunits in vitro. The regulation of this enzyme activity is of interest because of the wide variety of agents that activate the kinase including serum, oncogene products, and phorbol ester in cultured cells (9)(10)(11) and insulin and progesterone in Xenopus oocytes (12,13). Although these agents initially act through different receptors, the pathways apparently converge to activate a single enzyme or a limited number of distinct enzymes.…”

“…Immunocomplexes were adsorbed to protein A-containing Staphylococcus aureus, and the bacteria immunocomplexes were washed several times with buffer A and with 1 M NaCl/10 mM Tris HCI, pH 7.2/0.1% Nonidet P-40. Immunoprecipitated proteins were solubilized by boiling for 2 min in electrophoresis sample buffer, resolved by NaDodSO4/PAGE, and visualized by autoradiography (13).…”

UVA Induces Ser381 Phosphorylation of p90RSK/MAPKAP-K1 via ERK and JNK Pathways

“…RSK is activated in response to a broad range of cellular perturbations (16,32,33), including oncogenic transformation (32), insulin (17,32), growth factors (33), phorbol esters (16,33), growth hormone, changes in cAMP levels, heat shock, ionizing radiation, and T cell receptor activation (16,17,32,33). The p90 RSK signal transduction pathways are involved in cell growth, proliferation, differentiation, and apoptotic death (16) (29), but this antibody shows some cross-reactivity with RSK2 and RSK3 when phosphorylated at the homologous serine.…”

Phospho-Signaling at Oocyte Maturation and Fertilization: Set Up for Embryogenesis and Beyond Part I. Protein Kinases

“…Some S6Ks have been identified and characterized for example in progesterone-and insulin-treated Xenopus eggs termed S6K II (S6K II, p92) different from S6K I (Erikson et al 1987), differential role in Xenopus embryogenesis (S6K; p70) (Schwab et al 1999), in Rana oocytes (S6K; p83) (Byun et al 2002), in porcine oocytes (Sugiura et al 2002), and G1 phase after completion of meiosis II in starfish unfertilized eggs (Mori et al 2006) but not in mouse oocytes (Dumont et al 2005). S6K (p90 Rsk ) inhibits the degradation of cyclin B by anaphase-promoting complex/cyclosome (APC/C) and results the second meiotic metaphase arrest .…”