Abstract
Protein kinase activity toward the 40 S ribosomal protein S6 is activated 6-fold in regenerating rat liver following 70% hepatectomy. The kinase is maximally activated within 2 h after surgery, remains active up to 36 h after surgery, and declines rapidly thereafter. The post-hepatectomy S6 kinase activity exhibits structural and functional similarity to an insulin-stimulated S6 kinase in H4 hepatoma cells. Both S6 kinase activities are cAMP- and Ca2+-independent, and have a requirement for [ethylenebis(oxyethylenenitrilo)]tetraacetic acid. The regenerating liver and the insulin-stimulated H4 hepatoma S6 kinase elute at similar positions when sequentially fractionated by anion-exchange and cation-exchange chromatography. Both enzymes migrate at Mr 70,000 on fast protein liquid chromatography Superose 12 gel filtration. In H4 hepatoma cells, activation of S6 kinase activity is reversed by removal of insulin, and the cells can then be restimulated. Freshly isolated hepatocytes from normal animals show low levels of S6 kinase activity which can be stimulated by epidermal growth factor and insulin. Hepatocytes prepared from regenerating liver remnant have constitutively high levels of S6 kinase activity, which is unresponsive to insulin plus epidermal growth factor and which remains elevated at least 2 h in the absence of exogenously added growth factors. These findings demonstrate S6 protein kinase activation in vivo, in the setting of regulated cell growth; as in cultured cells, activation of S6 kinase probably represents an early step in the pleiotypic response elicited by activation of growth factor receptors.
Highlights
Protein kinase activitv toward the 40 S ribosomal have been shown to contain an activatedS6 protein S6 is activated6:fold in regenerating rat liver protein kinase [11-14]
The kinase is maximally for S6 and is independent of CAMP and Ca2+; it is distinct activated within 2 h after surgery, remains activeup from a variety of protein kinases [15-17] which can phosphoto 36 h after surgery, anddeclines rapidly thereafter. rylate S6 in uitros,uch as protein kinaseC, protein kinase A, The post-hepatectomy S6 kinaseactivityexhibits protease-activated kinase 2 or an histone H4 protein kinase
With regard to the initiatingsignals, sevtivity, which is unresponsive to insulin plus epidermal eral lines of investigation have led to the concept thaotne or growth factor andwhich remains elevatedat least 2 h more growth factors, delivered primarily via the portalcircuin the absence of exogenously added growth factors. lation, are of importance
Summary
In homogenization buffer (5 ml) andhomogenized with 25 strokes in a Dounce homogenizer. Cytoplasmic extracts from livers of partially hepatectomized, sham-operated, or normal fed rats were assayed for S6 protein kinase activity using rat liver 40 Sribosomal subunits as exogenous substrate. Rat liver 40 S ribosomal subunits were prepared as in Ref. 12. By 2 h after surgery, there is a 6-fold increase in S6 kinase activity compared to extracts prepared from either sham-operatedrats or normal rats. 60 p~ ATP (specific activity 500-3,000 cpm/pmol),and 0.44 A260 parison, DNA synthesis in regenerating liver first increases units of 40 S ribosomal subunits. Increase in overall protein kinase activity, but is specific for S6
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