Activation Of S6 Protein Kinase Research Articles

The Human Papillomavirus Type 16 E6 Oncoprotein Activates mTORC1 Signaling and Increases Protein Synthesis

The mammalian target of rapamycin (mTOR) kinase acts as a cellular rheostat that integrates signals from a variety of cellular signal transduction pathways that sense growth factor and nutrient availability as well as intracellular energy status. It was previously reported that the human papillomavirus type 16 (HPV16) E6 oncoprotein may activate the S6 protein kinase (S6K) through binding and E6AP-mediated degradation of the mTOR inhibitor tuberous sclerosis complex 2 (TSC2) (Z. Lu, X. Hu, Y. Li, L. Zheng, Y. Zhou, H. Jiang, T. Ning, Z. Basang, C. Zhang, and Y. Ke, J. Biol. Chem. 279:35664-35670, 2004; L. Zheng, H. Ding, Z. Lu, Y. Li, Y. Pan, T. Ning, and Y. Ke, Genes Cells 13:285-294, 2008). Our results confirmed that HPV16 E6 expression causes an increase in mTORC1 activity through enhanced phosphorylation of mTOR and activation of downstream signaling pathways S6K and eukaryotic initiation factor binding protein 1 (4E-BP1). However, we did not detect a decrease in TSC2 levels in HPV16 E6-expressing cells. We discovered, however, that HPV16 E6 expression causes AKT activation through the upstream kinases PDK1 and mTORC2 under conditions of nutrient deprivation. We show that HPV16 E6 expression causes an increase in protein synthesis by enhancing translation initiation complex assembly at the 5' mRNA cap and an increase in cap-dependent translation. The increase in cap-dependent translation likely results from HPV16 E6-induced AKT/mTORC1 activation, as the assembly of the translation initiation complex and cap-dependent translation are rapamycin sensitive. Lastly, coexpression of the HPV16 E6 and E7 oncoproteins does not affect HPV16 E6-induced activation of mTORC1 and cap-dependent translation. HPV16 E6-mediated activation of mTORC1 signaling and cap-dependent translation may be a mechanism to promote viral replication under conditions of limited nutrient supply in differentiated, HPV oncoprotein-expressing proliferating cells.

Insulin-induced Drosophila S6 kinase activation requires phosphoinositide 3-kinase and protein kinase B.

An important mechanism by which insulin regulates cell growth and protein synthesis is through activation of the p70 ribosomal S6 protein kinase (S6K). In mammalian cells, insulin-induced PI3K (phosphoinositide 3-kinase) activation, generates the lipid second messenger PtdIns(3,4,5) P (3), which is thought to play a key role in triggering the activation of S6K. Although the major components of the insulin-signalling pathway are conserved in Drosophila, recent studies suggested that S6K activation does not require PI3K in this system. To investigate further the role of dPI3K (Drosophila PI3K) in dS6K (Drosophila S6K) activation, we examined the effect of two structurally distinct PI3K inhibitors on insulin-induced dS6K activation in Kc167 and S2 Drosophila cell lines. We found that both inhibitors prevented insulin-stimulated phosphorylation and activation of dS6K. To investigate further the role of the dPI3K pathway in regulating dS6K activation, we also used dsRNAi (double-stranded RNA-mediated interference) to decrease expression of dPI3K and the PtdIns(3,4,5) P (3) phosphatase dPTEN ( Drosophila phosphatase and tensin homologue deleted on chromosome 10) in Kc167 and S2 cells. Knock-down of dPI3K prevented dS6K activation, whereas knock-down of dPTEN, which would be expected to increase PtdIns(3,4,5) P (3) levels, stimulated dS6K activity. Moreover, when the expression of the dPI3K target, dPKB (Drosophila protein kinase B), was decreased to undetectable levels, we found that insulin could no longer trigger dS6K activation. This observation provides the first direct demonstration that dPKB is required for insulin-stimulated dS6K activation. We also present evidence that the amino-acid-induced activation of dS6K in the absence of insulin, thought to be mediated by dTOR (Drosophila target of rapamycin), which is unaffected by the inhibition of dPI3K by wortmannin. The results of the present study support the view that, in Drosophila cells, dPI3K and dPKB, as well dTOR, are required for the activation of dS6K by insulin.

Activation of ribosomal S6 kinase (RSK) during porcine oocyte maturation.

The normal kinetics of ribosomal S6 kinase (RSK) during the meiotic maturation of porcine oocytes were examined. The phosphorylation states of RSK and extracellular signal-regulated kinase (ERK), major mitogen-activated protein (MAP) kinases in maturating porcine oocytes, were detected by Western blotting analysis. The S6 protein kinase activity was assayed using a specific substrate peptide which contained the major phosphorylation sites of S6 kinase. Full phosphorylation of RSK was correlated with ERK phosphorylation and was observed before germinal vesicle breakdown. S6 kinase activity was low in both freshly isolated and 20 h cultured oocytes. S6 kinase activity was significantly elevated in matured oocytes to a level about 6 times higher than that in freshly isolated oocytes. Furthermore, full phosphorylation of RSK was inhibited when oocytes were treated with U0126, a specific MAP kinase kinase inhibitor, in dose-dependent manner, indicating that RSK is one of the substrates of MAP kinase. These results suggest that the activation of RSK is involved in the regulation of meiotic maturation of porcine oocytes.

Molecular structure of a major insulin/mitogen-activated 70-kDa S6 protein kinase.

The molecular structure of a rat hepatoma 70-kDa insulin/mitogen-stimulated S6 protein kinase, obtained by molecular cloning, is compared to that of a rat homolog of the 85-kDa Xenopus S6 protein kinase alpha; both kinases were cloned from H4 hepatoma cDNA libraries. The 70-kDa S6 kinase (calculated molecular mass of 59,186 Da) exhibits a single catalytic domain that is most closely related in amino acid sequence (56% identity) to the amino-terminal, kinase C-like domain of the rat p85 S6 kinase (calculated molecular mass of 82,695 Da); strong similarity extends through a further 67 residues carboxyl-terminal to the catalytic domain (40% identity), corresponding to a region also conserved among the kinase C family. Outside of this segment of approximately 330 amino acids, the structures of the p70 and p85 S6 kinases diverge substantially. The p70 S6 kinase is known to be activated through serine/threonine phosphorylation by unidentified insulin/mitogen-activated protein kinases. A model for the regulation of p70 S6 protein kinase activity is proposed wherein the low activity of the unphosphorylated enzyme results from the binding of a basic, inhibitory pseudosubstrate site (located carboxyl-terminal to the extended catalytic domain) to an acidic substrate binding region (located amino-terminal to the catalytic domain); substrate binding is thereby prevented. S6 kinase activation requires displacement of this inhibitory segment, which is proposed to occur consequent to its multiple phosphorylation. The putative autoinhibitory segment contains several serine and threonine residues, each followed directly by a proline residue. This motif may prevent autophosphorylation but permit transphosphorylation; two of these serine residues reside in a maturation promoting factor (MPF)/cdc-2 consensus motif. Thus, hormonal regulation of S6 kinase may involve the action of MPF/cdc-2 or protein kinases with related substrate specificity.

Evidence for insulin-dependent activation of S6 and microtubule-associated protein-2 kinases via a human insulin receptor/v-ros hybrid.

The abilities of a series of six mutants of the human insulin receptor, an insulin receptor/v-ros hybrid (IR-ros) and the P68gag-ros transforming protein to stimulate S6 protein kinase have been assessed. Insulin receptor mutants in which either 1 or 2 tyrosine residues have been replaced with phenylalanine (YF1, YF3) have lost some or all of the capacity to mediate the activation of S6 kinase in response to insulin. None of the four mutants that contain deletions (spBam, spBamYF3, iBgl, T-t) elicit an insulin-dependent stimulation of S6 kinase. A previous study of the IRros hybrid receptor demonstrated that it was unable to cause either insulin-stimulated thymidine incorporation or glucose uptake (Ellis, L., Morgan, D. O., Jong, S.-M., Wang, L.-H., Roth, R. A., and Rutter, W. J. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 5101-5105). In contrast, the IRros chimera appears to mediate the activation of S6 protein kinase by insulin. In further evaluating the biological activities of the IRros hybrid, we have examined its effects on a microtubule-associated protein-2 (MAP2) kinase that is thought to be an early target in the cascade of reactions leading to increased S6 phosphorylation (Sturgill, T. W., Ray, L. B., Erickson, E., and Maller, J. L. (1988) Nature 334, 715-718). We find that the IRros receptor stimulates the MAP2 protein kinase from 3- to 6-fold in insulin-treated cells, conferring more than a 30-fold increase in the insulin sensitivity of MAP2 kinase activation.

Stimulation of ribosomal protein S6 kinase activity by pp60v-src or by serum: dissociation from phorbol ester-stimulated activity.

Ribosomal protein S6 kinase activity was measured in lysates prepared from serum-deprived chicken embryo fibroblasts (CEF) treated for various times with phorbol 12-myristate 13-acetate (PMA). Maximal activity was observed within 15 min, and it declined to the initial level by 4 hr. Incubation of these cells with PMA 4-60 hr after the initial treatment did not result in an additional increase in S6 protein kinase activity. These results are consistent with down-regulation of the PMA receptor, protein kinase C, and the dependence of PMA-stimulated S6 kinase activity on this enzyme. Long-term pretreatment of CEF with PMA only partially attenuated the stimulation of the S6 protein kinase activity by serum or by expression of the Rous sarcoma virus transforming gene product, pp60v-src. A similar protein kinase activity also was stimulated in cells treated with cycloheximide or sodium vanadate. Pretreatment with PMA had little effect on this response. These data indicate that it is likely that there are at least two mechanisms through which S6 kinase activity can be regulated, one of which apparently utilizes protein kinase C whereas the other(s) does not. Additional experiments show PMA-stimulated glucose transport was not attenuated by long-term incubation with phorbol ester, suggesting that another mechanism, which is not dependent on the presence of protein kinase C, maintains this response after the proposed down-regulation of the PMA receptor.

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.

Activation of a ribosomal protein S6 protein kinase in Xenopus oocytes by insulin and insulin-receptor kinase.

Previous studies in this laboratory have shown that insulin treatment of Xenopus oocytes leads to an increase in phosphorylation of ribosomal protein S6. To investigate the mechanism of this increase, S6 kinase activity was measured in lysates of oocytes exposed to insulin. Insulin caused a rapid 4- to 6-fold increase in S6 kinase activity, which was maximal by 20 min and which could be reversed by removal of insulin prior to homogenization. Dose-response curves showed a detectable increase in specific activity at 1 nM insulin with a maximal effect at 100 nM. Treatment of oocytes with puromycin did not prevent this increase in S6 kinase activity, suggesting activation rather than synthesis of the enzyme. DEAE-Sephacel chromatography of extracts from insulin-treated oocytes revealed two peaks of S6 kinase activity, and the specific activity of the peak eluting at 300 nM NaCl was increased 3-fold in oocytes treated with insulin. The same peak of S6 kinase activity was increased 40% within 10 min in oocytes injected with highly purified insulin-receptor kinase. These results indicate that the insulin-dependent increase in S6 phosphorylation is due, at least in part, to activation of an S6 protein kinase, and this activation may result from the action of the insulin receptor at an intracellular location.