Mitogen‐responsive S6 kinase

Abstract

Many cell lines respond to mitogenic stimuli (serum, growth factors) with rapid phosphorylation of the ribosomal protein S6 at several serine sites. We have tried to identify the protein kinase(s) mediating this effect of growth stimuli. Examining post-DEAE chromatography fractions of S49 kin ~ cell extracts, we could detect a highly active effector-independent S6 kinase with specificity for serine residues. The study was extended to the presumably homologous human enzyme, using HeLa S3 cells as model system. Activity yields increased up to sevenfold when exhausted HeLa cells were supplied with fresh medium plus serum. The enzyme uses ATP, not GTP, as cosubstrate, 40-S or 80-S (reassociated from subunits) ribosomal particles being substrate. The optimal K + concentration, measured at 3 mM Mg2+, is 35 mM. Under optimized assay conditions S6 phosphorylation proceeded faster in vitro than it appeared to do in vivo. The apparent M , of the enzyme, as estimated by gel filtration on Sephadex G-100, is 56000 (determination in the presence of 200 mM KCI in 25 mM phosphate buffer). Tighter binding to DEAE-Sephacel and higher specificity for S6 distinguishes this enzyme from the following S6-phosphorylating protein kinases: protein kinase C, protease-activated kinase 11, histone-4 phosphotransferase and an enzyme with the properties of casein kinase I. In published summaries of observations shown here and in a follow-up study with chick embryo fibroblasts, the enzyme(s) has been referred to as mitogenresponsive S6 kinase(s) [Martini, 0. H. W. and Lawen, A. (1985) [14, 351, and fertilization of oocytes [16]. Serum-induced S6 phosphorylation was promptly reversed when cells were exposed to stress, a condition that also causes inhibition of translation [17].As an end-point of signalling pathways, S6 can be utilized, in analogy to phosphorylase [IS], to unravel signal flux by proceeding from distal to proximal. Towards this goal we have sought the S6-phosphorylating kinase(s) mediating growth- Abbreviations. PKC, CaZ +-phosphatidylserine-diolein-dependent protein kinase (protein kinase C); CAMP-PK, CAMP-dependent protein kinase; PAK 11, protease-activated protein kinase 11; histone-4 PK, histonc-4 phosphotransferase; S6-KI1, Xenopus oocyte S6 kinase I1 (two closely related proteins referred to as SG-KIIa and S6-KIIP); NaCI/Pi, phosphate-buffered salinc.Enzyme. Protein kinases (EC 2.7.1.37). promoting signals. To obviate possible interference by CAMPdependent protein kinase (CAMP-PK) (the enzyme has been implicated in both negative, antagonizing anabolic signals [19], and positive [20 -241 control of S6 phosphorylation), the search was begun in S49 kin-murine lymphoma cells which express CAMP-PK at a subnormal level [25]. A strong S6-phosphorylating activity was found, which was effector-independent and chromatographically distinct from several effector-dependent Sh-phosphorylating kinases of broader substrate range. A similar human enzyme(s) was then detected using HeLa S3 cells, extending the pioneering work of McConkey an...

Molecular Cloning, Developmental Expression, and Phosphorylation of Ribosomal Protein S6 in the Endocrine Gland Responsible for Insect Molting

“…Phosphoamino acid analysis revealed that S6 was phosphorylated exclusively at the serine residues (Fig. 3b), a result consistent with all past reports on the subject (34).…”