Activation of S6 kinase in astroglial cells by FGFa and FGFb.

Basic (b) and acidic (a) forms of the fibroblast growth factor (FGF) promoted a rapid increase of the cytosolic S6 kinase activity in astroglial cells. S6 kinase activity was maximal 10 min after addition of the factors to cell cultures and remained at this level for at least 30 min. Half-activation of the enzyme was obtained with 3 ng/ml FGFa. Heparin (100 micrograms/ml) potentiated the response to suboptimal concentrations of FGFa. This growth factor appeared to stimulate an astroglial S6 kinase resembling that stimulated by insulin, IGF1, TPA and cAMP. Although FGFb is more potent than FGFa in stimulating proliferation of Chinese hamster lung fibroblasts (CCL39), it was not more efficient than FGFa in stimulating the S6 kinase activity of astroglial cells.

[A model for studying the transmission of information produced by certain growth factors: activation mechanisms of S6 kinase in cultured astrocytes]. / Un modèle pour l'étude de la transmission de l'information produite par certains facteurs de croissance: les mécanismes d'activation de la S6 kinase dans les astrocytes en culture.

Treatment of cultured astrocytes from 2-day-old rat cerebral hemispheres with insulin, somatomedin C (IGF1), thrombin and acidic or basic fibroblast growth factors promoted a rapid activation of a cytosolic protein kinase (S6 kinase) which phosphorylates ribosomal protein S6. The phorbol ester (TPA) also triggered a rapid increase in S6 kinase activity. Two agonists of adenylate cyclase activity (forskolin and isoproterenol) and the cyclic AMP analog (dibutyryl cAMP) also stimulated the same S6 kinase. These observations support the idea that several pathways might promote the activation of the same entity that is regarded as one of the primary targets of signals elicited by growth factors.

Properties of the 12-O-tetradecanoylphorbol-13-acetate-stimulated S6 kinase from rat astroglial cells.

The S6 kinase activity of astroglial cells in primary culture stimulated by 12-O-tetradecanoylphorbol-13-acetate (TPA) has been studied. This activity was eluted as a single peak at 0.15 M NaCl from a DEAE-Sephacel column. The chromatography of this peak on phosphocellulose revealed an activity eluted at 0.15 M NaCl. This partially purified enzyme had a sedimentation coefficient of 3.7S; Km values were 2 X 10(-5) M for ATP and 10(-6) M for 40S ribosomal subunits. The optimal Mg2+ concentration requirement was 2-3 mM. Mn2+ and Co2+ could substitute for Mg2+ (optimum concentrations 1.5 and 0.8 mM, respectively), but these cations were strong inhibitors in the presence of Mg2+. The enzyme was inhibited by N-ethylmaleimide, indicating that it contained thiol groups. This S6 kinase used ATP, but not GTP, as a phosphate donor, and exhibited great specificity for S6 as phosphate acceptor. Whole histones and protamine were slightly phosphorylated whereas phosvitin, histone H1, and surprisingly the peptide Arg-Arg-Leu-Ser-Ser-Leu-Arg-Ala were not phosphorylated. The TPA-stimulated S6 kinase resembles the insulin-, fibroblast growth factor- and cyclic AMP-stimulated enzymes, suggesting that several pathways might activate the same entity.

Insulin and insulin-like growth factor 1 receptors during postnatal development of rat brain.

The binding of insulin and insulin-like growth factor 1 (IGF1) to high-affinity sites in the brain of rats aged 2-37 days was studied. Specific binding of insulin and IGF1 was assessed using tracer concentrations of 125I-insulin or 125I-IGF1. Sites for insulin and IGF1 were distinguished in these conditions as shown by competition experiments. The Kd were 3.6 nM (insulin) and 2.0 nM (IGF1). These values did not change significantly over the age range studied. The numbers of high-affinity binding sites for insulin and IGF1 were similar in adult animals. IGF1 binding was higher than the insulin binding in 2-day-old animals. The binding capacity for both insulin and IGF1 decreased from birth to age 15 and days remained stable thereafter. Tyrosine kinase activity, which is associated with these receptors, was measured using the artificial substrate poly (Glu, Tyr). It decreased over the first 15 days of life and remained stable thereafter. Autophosphorylation of the receptors confirmed this result. This decrease appears to be due to changes in the numbers of the two types of receptors, and is probably a reflection mainly of the variation in the number of IGF1 receptors. Similar results for insulin and IGF1 binding as well as tyrosine kinase activity were obtained with hypothyroid rats.

Activation of an S6 kinase from rat astroglial cells by cAMP.

Forskolin and isoproterenol, agonists of adenylate cyclase activity, and dibutyryl cyclic AMP, stimulated an S6 kinase activity in astroglial cells. This activity was insensitive to the thermostable inhibitor of cyclic AMP-dependent protein kinase and had the same behaviour on a DEAE-Sephacel column as the mitogen stimulated S6 kinase. These observations support the idea that the cyclic AMP cascade, as well as various growth factors, can activate S6 kinase.