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

Abstract

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...

Mitogen‐responsive S6 kinase

“…Although the PAK-11-and histone-4 PK-centered variants of the 'fragment hypothesis ' have not yet been invalidated, 'S6 kinases' have meanwhile been described for a variety of systems: Xenopus oocytes [38-401, 3T3 cells [41,421, chick embryo fibroblasts [43, 441, hamster fibroblasts [45], murine fibroblast-melanoma hybrid cells (461, sea star oocytes [47] and bovine liver [48] (for homology speculation see Discussion ; for insulin-responsive activity see [9]), S6 kinase activities could be modulated in vitro by phosphataselkinase action [49,501. Furthermore, at least for Xenopus oocyte S6 kinase 11, sequence data unambiguously show that it is not a form of PKC [51].…”