Stimulated S6 Kinase Research Articles

Biochemical and functional characterizations of small GTPase Rheb and TSC2 GAP activity.

Tuberous sclerosis complex (TSC) is a genetic disease caused by a mutation in either the tsc1 or tsc2 tumor suppressor gene. Recent studies have demonstrated that TSC2 displays GAP (GTPase-activating protein) activity specifically towards the small G protein Rheb and inhibits its ability to stimulate the mTOR signaling pathway. Rheb and TSC2 comprise a unique pair of GTPase and GAP, because Rheb has high basal GTP levels and TSC2 does not have the catalytic arginine finger found in Ras-GAP. To investigate the function of TSC2 and Rheb in mTOR signaling, we analyzed the TSC2-stimulated Rheb GTPase activity. We found that Arg15, a residue equivalent to Gly12 in Ras, is important for Rheb to function as a substrate for TSC2 GAP. In addition, we identified asparagine residues essential for TSC2 GAP activity. We demonstrated a novel catalytic mechanism of the TSC2 GAP and Rheb that TSC2 uses a catalytic "asparagine thumb" instead of the arginine finger found in Ras-GAP. Furthermore, we discovered that farnesylation and membrane localization of Rheb is not essential for Rheb to stimulate S6 kinase (S6K) phosphorylation. Analysis of TSC1 binding defective mutants of TSC2 shows that TSC1 is not required for the TSC2 GAP activity but may function as a regulatory component in the TSC1/TSC2 complex. Our data further demonstrate that GAP activity is essential for the cellular function of TSC2 to inhibit S6K phosphorylation.

Identification of the S6 kinase activity stimulated in quiescent brine shrimp embryos upon entry to preemergence development as p70 ribosomal protein S6 kinase: Isolation ofArtemia franciscanap70S6kcDNA

We previously demonstrated that a protein kinase responsible for phosphorylating 40S ribosomal subunits is activated in quiescent Artemia franciscana embryos within 15 min of restoration of normal tonicity and incubation at 30°C. Here, we identify the activated S6 kinase as A. franciscana p70 ribosomal S6 kinase (p70S6k) subsequent to the isolation of an Artemia p70S6kcDNA. The protein conceptually translated from cDNA has 70% similarity and 64% identity to both Drosophila melanogaster and human p70S6k. Southern blot analysis is consistent with presence of a single p70S6kgene. Two transcripts of 5.4 and 2.7 kb were found. Abundance of both mRNAs increased dramatically around 4 h of preemergence development, and exhibited different steady-state level variation thereafter. Stimulated S6 kinase activity, partially purified by Superose 6 chromatography, correlated best with the slowest migrating, ~65 kDa, form detected by Western analysis using a specific polyclonal antibody made to a peptide from the predicted p70S6kNH2-terminus. Furthermore, the A. franciscana p70S6kwas immunoprecipitated with the same antibody, showing in parallel an S6 kinase activity similar to peak profiles. We conclude that the stimulated S6 kinase activity is that of an ortholog of human p70S6kthat may be involved in the regulation of protein synthesis during preemergence development in A. franciscana species.Key Words: p70 ribosomal S6 kinase cDNA, brine shrimp, development.

Identification of the S6 kinase activity stimulated in quiescent brine shrimp embryos upon entry to preemergence development as p70 ribosomal protein S6 kinase: Isolation of <i>Artemia franciscana</i> p70<sup>S6k</sup> cDNA

We previously demonstrated that a protein kinase responsible for phosphorylating 40S ribosomal subunits is activated in quiescent Artemia franciscana embryos within 15 min of restoration of normal tonicity and incubation at 30 degrees C. Here, we identify the activated S6 kinase as A. franciscana p70 ribosomal S6 kinase (p70S6k) subsequent to the isolation of an Artemia p70S6k cDNA. The protein conceptually translated from cDNA has 70% similarity and 64% identity to both Drosophila melanogaster and human p70S6k. Southern blot analysis is consistent with presence of a single p70S6k gene. Two transcripts of 5.4 and 2.7 kb were found. Abundance of both mRNAs increased dramatically around 4 h of preemergence development, and exhibited different steady-state level variation thereafter. Stimulated S6 kinase activity, partially purified by Superose 6 chromatography, correlated best with the slowest migrating, approximately 65 kDa, form detected by Western analysis using a specific polyclonal antibody made to a peptide from the predicted p70S6k NH2-terminus. Furthermore, the A. franciscana p70S6k was immunoprecipitated with the same antibody, showing in parallel an S6 kinase activity similar to peak profiles. We conclude that the stimulated S6 kinase activity is that of an ortholog of human p70S6k that may be involved in the regulation of protein synthesis during preemergence development in A. franciscana species.

Identification of a Novel Ca2+-Stimulated S6-Kinase in Rat Liver

Extracellular calcium addition transiently stimulated two S6 peptide kinase activities in isolated rat hepatocytes. Mono Q chromatography revealed that the activities eluting at 0.15 M NaCl and 0.18 M NaCl were stimulated 4-fold and 2-fold, respectively. The kinase stimulated by calcium was a 40000-Mr S6 peptide kinase, as demonstrated by partial purification from whole liver. The protein kinase did not crossreact with antibodies directed against the N- or C-terminal part of p70 ribosomal S6 kinase (p70S6K) and the C-terminal part of p90 ribosomal S6 kinase (p90rsk). Following digestion of 40000-Mr S6 peptide kinase with trypsin, six peptides were sequenced. There was no similarity with the sequences of p70S6Kand p90rsk. Moreover, the obtained sequences could not be identified in the SwissProt or EMBL-genebank databases, suggesting that 40000-Mr S6 peptide kinase probably represents a novel protein kinase.

Effect of rapamycin on prolactin-stimulated S6 kinase activity and milk product formation in mouse mammary explants

The in vitro effects of rapamycin on prolactin (PRL)-stimulated S6 kinase activity and milk product synthesis were investigated in cultured mouse mammary tissues. Mouse mammary gland explants were initially incubated for 24 h in M199 media containing 1 μg/ml insulin and 10 −7 M cortisol. A subsequent treatment of the tissues with 1 μg/ml PRL for 12 h caused a 98% increase in S6 kinase activity in the cytosolic fraction; effects were not observed at earlier times. PRL at or above 500 ng/ml was needed to elicit a maximum stimulation of S6 kinase activity, and this response was specific for lactogenic hormones (PRL, hPL, hGH). Rapamycin, an inhibitor of 70 K S6 kinase, was employed to assess the possible physiological role of S6 kinase in the PRL stimulation of milk product formation. Rapamycin (25–100 ng/ml) impeded, in a dose-dependent fashion, the PRL stimulation of casein, lipid and lactose synthesis in concert with its inhibition of cytoplasmic S6 kinase activity. These results suggest a possible role for the activation of 70 K kinase in the signalling pathway for the PRL regulation of milk product synthesis in the mammary gland.

Insulin signaling and its regulation of system A amino acid uptake in cultured rat vascular smooth muscle cells.

Hyperinsulinemia has been recognized as an independent risk factor for atherosclerosis. However, its exact mechanisms are still unclear. In our previous work, we showed that 10 nmol/L insulin stimulated neither mitogen-activated protein kinase (MAP kinase) activity nor [3H]thymidine incorporation but did stimulated S6 kinase through the specific insulin receptors in cultured rat vascular smooth muscle cells (VSMCs). In this study, we observed that > or = 1 nmol/L insulin stimulated tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and activated IRS-1-dependent phosphatidylinositol 3'-kinase (PI 3'-kinase) and p70 S6 kinase (p70S6K) but not MAP kinase (extracellular signal-regulated kinase 2) and p90 S6 kinase (p90RSK). However, 10 nmol/L insulin-like growth factor I stimulated all these pathways. Finally, 10 nmol/L insulin stimulated alpha-amino-isobutyric acid (AIB) uptake, and wortmannin (100 nmol/L) completely inhibited insulin-stimulated AIB uptake, whereas rapamycin (20 nmol/L) had no such effect. Furthermore, cycloheximide (10 micrograms/mL) completely inhibited insulin-stimulated AIB uptake, but actinomycin D (5 micrograms/mL) failed to inhibit this. Thus, we reached the following conclusions: (1) Insulin (1 nmol/L) induced phosphorylation of IRS-1 and activated the PI 3'-kinase and p70S6K pathways in VSMCs, even though 10 nmol/L insulin did not significantly stimulate MAP kinase or p90RSK. (2) Stimulation of AIB uptake by insulin was regulated at the translational level via wortmannin-sensitive pathways but not p70S6K pathways.

Phosphatidylinositol 3-Kinase Activity Is Required for Hepatocyte Growth Factor-induced Mitogenic Signals in Epithelial Cells

Phosphatidylinositol (PI) 3-kinase is an important enzyme implicated in growth factor-stimulated intracellular signaling. In this study we have shown that hepatocyte growth factor (HGF) induces a rapid tyrosine phosphorylation of PI 3-kinase and association with HGF receptor/Met in Mv1Lu epithelial cells. Murine mammary carcinoma (SP1) cells, which co-express HGF and HGF receptor/Met, showed sustained phosphorylation of PI 3-kinase. Wortmannin, a potent inhibitor of PI 3-kinase, inhibited HGF-induced PI 3-kinase activity, proliferation of Mv1Lu cells, and spontaneous growth of SP1 cells in a dose-, and time-dependent manner. Transfection of a dominant negative mutant p85 (Deltap85) subunit of PI 3-kinase into SP1 cells strongly inhibited HGF-stimulated proliferation and PI 3-kinase activity. However, wortmannin did not influence HGF-induced c-Jun expression. Furthermore, HGF stimulated S6 kinase activity, but its activity was not required for HGF-induced proliferation. Overall, these results suggest that HGF-induced PI 3-kinase activity is important for the mitogenic action of HGF in epithelial cells and further demonstrate that expression of c-Jun is not influenced by inhibition of PI 3-kinase activity.

Coordinate, biphasic activation of p44 mitogen-activated protein kinase and S6 kinase by growth factors in hamster fibroblasts. Evidence for thrombin-induced signals different from phosphoinositide turnover and adenylylcyclase inhibition.

In CCL39 cells transfected with m1-muscarinic receptors, carbachol stimulates phosphoinositide turnover and early events associated with mitogenesis as efficiently as thrombin but, in contrast to thrombin, fails to induce cell proliferation (Seuwen, K., Kahan, C., Hartmann, T., and Pouysségur, J. (1990) J. Biol. Chem. 265, 22292-22299). We show here that the action of the two agents can be dissociated at the level of S6 kinase and mitogen-activated protein kinase (MAP kinase) activation. Mitogenic concentrations of thrombin and basic FGF were found to stimulate S6 kinase activity, measured in whole cell lysates, with a biphasic time course; an early peak of activity is induced 10 min following stimulation and a sustained phase of activity can be measured over several hours. A very similar profile emerged for p44 MAP kinase (p44mapk), assayed in immunoprecipitates. In this case, the activity first peaks at 6-8 min, preceding S6 kinase. In contrast to thrombin and FGF, carbachol stimulates S6 kinase and MAP kinase only transiently, corresponding to the first peak of activity, but the sustained phase is not observed. Similarly, phorbol dibutyrate induces an early phase of activity only. Pertussis toxin (PTX), which is known to block thrombin mitogenicity efficiently, inhibited the first peak of thrombin-induced S6 kinase and MAP kinase activity only partially, but totally blocked the sustained phase. The toxin had no effect on FGF-induced kinase activities. The cAMP elevating hormone PGE1 did not inhibit p44mapk or S6 kinase activation by thrombin or FGF, demonstrating that the PTX-sensitive signal generated by thrombin does not depend on a Gi-mediated sustained inhibition of adenylylcyclase. Surprisingly, PGE1 was found to stimulate sustained phase S6 kinase activity both alone and in synergy with FGF or thrombin. This result, as well as the biphasic activation of S6 kinase by thrombin, could be qualitatively reproduced in immunocomplex kinase assays using an antiserum immunoprecipitating p70 S6 kinase (p70S6k). Our data show that activation of phosphoinositide turnover and PKC does not quantitatively explain thrombin action, in particular the sustained phase of kinase activities, which critically depends on a PTX-sensitive signal different from adenylylcyclase inhibition. We postulate that this signal does not exclusively originate from the recently identified G protein-coupled thrombin receptor.

Regulation of S6 kinase activity in renal proximal tubule.

The proximal tubule undergoes hypertrophy in response to loss of functioning renal mass and hyperplasia following injury by ischemia or nephrotoxins. Both hypertrophic growth and cell proliferation are characterized by increases in the rate of protein synthesis. To investigate regulation of protein synthesis in mammalian proximal tubule cells, potential peptide mediators of proximal tubule growth, epidermal growth factor (EGF) and angiotensin II, were studied in cultured rabbit proximal tubule cells. Although only EGF stimulated DNA synthesis, both agonists stimulated protein synthesis. One potential regulatory mechanism of eukaryotic protein synthesis involves phosphorylation of ribosomal protein S6 by activation of a specific serine/threonine kinase (S6 kinase). Both EGF and angiotensin II stimulated S6 kinase activity and S6 phosphorylation. Phorbol 12-myristate 13-acetate was also found to activate S6 kinase, and 24 h of pretreatment to deplete protein kinase C inhibited subsequent S6 kinase activation by a high concentration (10(-6) M) of angiotensin II. To determine whether S6 kinase was also activated in the kidney in vivo, S6 kinase activity was examined after ablation of renal mass. Within 1 h after contralateral nephrectomy, S6 kinase activity increased in rat renal cortex. In summary, both EGF and angiotensin II stimulated protein synthesis and S6 kinase activity in cultured proximal tubule cells, and S6 kinase activity also increased in renal cortex after contralateral nephrectomy.

Intracellular signaling by a mutant human insulin receptor lacking the carboxyl-terminal tyrosine autophosphorylation sites.

We have recently characterized a mutant insulin receptor (Y/F2) in which the two tyrosines in the carboxyl terminus (Tyr1316, Tyr1322) were mutated to phenylalanine. Compared with wild type receptors, the Y/F2 receptor exhibited markedly enhanced sensitivity to insulin-stimulated DNA synthesis with normal insulin-stimulated glucose uptake (Takata, Y., Webster, N. J. G., and Olefsky, J. M. (1991) J. Biol. Chem. 266, 9135-9139). In this paper, we present further evidence for the divergence of the metabolic and mitogenic signaling pathways utilized by the insulin receptor. The mutant receptor showed normal sensitivity and responsiveness for insulin-stimulated glucose incorporation into glycogen. The insulin sensitivity for phosphorylation of two substrates (pp180 and pp220) was the same in both Y/F2 cells and HIRc cells. Phosphotyrosine content, however, was greater in Y/F2 cells than in HIRc cells, especially in the basal state. Insulin stimulated S6 kinase activity 2-6-fold, with an ED50 of -10 nM in Rat 1 cells and 0.5 nM in HIRc cells. The sensitivity to insulin was enhanced in Y/F2 cells with an ED50 of 0.1 nM. These effects were insulin-specific, since insulin-like growth factor (IGF)-I-stimulated mitogenesis was normal. In summary: 1) Y/F2 receptors exhibit normal metabolic and enhanced mitogenic signaling; 2) the enhanced mitogenic signaling is specific for the insulin receptor in the Y/F2 cells, since IGF-I-stimulated mitogenesis is normal; 3) Y/F2 cells display increased endogenous substrate phosphorylation and augmented insulin-stimulated S6 kinase activity placing these responses among insulin's mitogenic effects; and 4) these results are consistent with the concept that the COOH-terminal tyrosine residues of the insulin receptor are normally inhibitory to mitogenic signaling.

Monoclonal antibody to the human insulin receptor, but not insulin, stimulates S6 kinase via human insulin receptors mutated at three major tyrosine autophosphorylation sites

Studies were carried out to examine the role of the major insulin receptor tyrosine autophosphorylation sites in stimulation of S6 kinase activity. For these studies, we employed HTC rat hepatoma cells transfected with and expressing human insulin receptors. In cells transfected with and expressing a large number of normal human insulin receptors (HTC-IR cells), the sensitivity of cells to insulin to stimulate S6 kinase was increased tenfold when compared to untransfected wild type HTC cells (HTC-WT cells). However, in cells transfected with and expressing a large number of mutated human insulin receptors where the tyrosines at three major autophosphorylation sites (1158, 1162, and 1163) were mutated to phenylalanines (HTC-F3 cells), there was no change in insulin sensitivity when compared to HTC-WT cells. We next studied the effect of a human-specific monoclonal antibody to the human insulin receptor, MA-5, on S6 kinase activation. In HTC-WT cells, MA-5 did not interact with endogenous rat insulin receptors and thus did not stimulate S6 kinase. In HTC-IR cells expressing normal human insulin receptors, MA-5 stimulated S6 kinase. Interestingly, MA-5, unlike insulin, was also able to stimulate S6 kinase in HTC-F3 cells expressing mutated receptors. In order to further understand the signaling mechanisms by MA-5 and insulin, two potential intermediate protein kinases were investigated. Neither insulin nor MA-5 appears to activate either microtubule-associated protein 2 (MAP-2) kinase or protein kinase C in these cells. These studies suggest therefore that: 1) insulin and MA-5 may signal S6 kinase activation by independent mechanisms that do not employ either MAP-2 kinase or protein kinase C; and 2) under certain circumstances, S6 kinase appears to be activated by mechanisms that are independent of insulin receptor tyrosine autophosphorylation.

Regulation of S6 kinase activity in Madin-Darby canine kidney renal epithelial cells.

Mitogenic stimulation of mammalian cells results in increased serine phosphorylation of ribosomal protein S6. Phorbol esters, which stimulate protein kinase C activity, can also increase S6 phosphorylation. In order to further investigate the role of protein kinase C in the activation S6 kinase, we studied the stimulation of an S6 kinase activity in response to phorbol ester and epinephrine in a renal epithelial cell line, Madin-Darby canine kidney cells (MDCK). In these cells, S6 phosphorylating activity in cytosolic extracts was increased following the addition of phorbol ester to the intact cells. S6 kinase and protein kinase C activities were measured in separate fractions prepared by DEAE-Sephacel fractionation of cytosolic extracts prepared from the same cells. The time course and dose-response curves for the effects of phorbol 12-myristate 13-acetate (PMA) on S6 kinase activity were similar to those for its effects on protein kinase C binding to the membrane fraction, indicating that S6 kinase activation was correlated with protein kinase C activation. Epinephrine, acting via alpha1-adrenergic receptors, also stimulated S6 kinase activity in MDCK cells; the magnitude of this effect was similar to that of PMA. However, epinephrine causes only a slight and transient association of protein kinase C with the membrane. The effect of epinephrine on S6 kinase activity, unlike that of PMA, was dependent on the presence of extracellular calcium. A23187, a calcium ionophore, could also stimulate S6 kinase activity. These results suggest that S6 kinase can be activated through more than one signaling pathway in MDCK cells. The properties of the PMA-stimulated S6 kinase were further investigated following partial purification of the enzyme. The S6 kinase was distinct from protein kinase C by several criteria. Noteably, the S6 kinase was highly specific for S6 as substrate. These results show that phorbol esters, acting through protein kinase C, stimulate the activity of a unique S6 kinase. This S6 kinase can also be activated through a signaling pathway that appears to be dependent on increased intracellular calcium.

A stimulated S6 kinase from rat liver: identity with the mitogen activated S6 kinase of 3T3 cells.

A number of approaches were tested for their ability to induce S6 phosphorylation and S6 kinase activation in rat liver, including i.p. injection of insulin, sodium orthovanadate or cycloheximide, as well as refeeding starved animals. All treatments led to increased S6 phosphorylation and activation of the apparent same enzyme. The most potent activator of the S6 kinase in liver extracts was cycloheximide. Maximum activation was achieved in 20 min at 1 mg cycloheximide/100 g body weight, with half-maximal activation in 10 min. Based on these findings a large-scale kinase purification procedure was established involving seven steps of chromatography. Following the final step a major protein band of Mr 70,000 was revealed. The protein was purified 20,000-fold, had a sp. act. of 640 nmol/min/mg of protein towards S6, autophosphorylated and was inactivated by phosphatase 2A. Peptide maps of autophosphorylated material were identical to those derived from the mitogen-activated kinase of 3T3 cells.

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.

A growth factor and TPA stimulated S6 kinase in cultured rat astroglial cells

A growth factor and TPA stimulated S6 kinase in cultured rat astroglial cells

An insulin-stimulated (ribosomal S6) protein kinase from soluble extracts of H4 hepatoma cells

Insulin stimulates the phosphorylation of the 40 S ribosomal subunit protein, S6, in intact 32P-labeled H4IIE-C3 cells, a rat hepatoma line. Cell-free cytosolic extracts from H4 cells exhibit a 5- to 10-fold increase in S6 protein kinase activity (measured by transfer of 32P to exogenous 40 S rat liver ribosomal subunits) when prepared from cells exposed to insulin prior to homogenization. Stimulation of S6 phosphorylation in intact cells and activation of S6 protein kinase in cell-free extracts are both detectable within 2 min after insulin, and are maximally stimulated by 10 min. Half-maximal stimulation is observed at 10 −11 m insulin. The stimulated S6 kinase activity requires ethylene glycol bis(β-aminoethyl ether)- N,N,N′,N′-tetraacetic acid to be present during the kinase assay for full expression. Despite the presence of a 5- to 10-fold increase in S6 protein kinase activity, the extracts from insulin-treated cells exhibit no stimulated kinase activity toward casein, histone, or ATP-citrate lyase assayed under the conditions employed for S6. Thus, insulin mediates the rapid activation of protein kinase specific for ribosomal protein S6 by an as yet unidentified mechanism.