Activation Of Ribosomal Protein S6 Kinase Research Articles

Quantitative proteomics and phosphoproteomics of PP2A-PPP2R5D variants reveal deregulation of RPS6 phosphorylation via converging signaling cascades

Genetic germline variants of PPP2R5D (encoding: phosphoprotein phosphatase 2 regulatory protein 5D) result in PPP2R5D-related disorder (Jordan's Syndrome), which is characterized by intellectual disability, hypotonia, seizures, macrocephaly, autism spectrum disorder, and delayed motor skill development. The disorder originates from de novo single nucleotide mutations, generating missense variants that act in a dominant manner. Pathogenic mutations altering 13 different amino acids have been identified, with the E198K variant accounting for ∼40% of reported cases. However, the generation of a heterozygous E198K variant cell line to study the molecular effects of the pathogenic mutation has been challenging. Here, we use CRISPR-PRIME genomic editing to introduce a transition (c.592G>A) in a single PPP2R5D allele in HEK293cells, generating E198K-heterozygous lines to complement existing E420K variant lines. We generate global protein and phosphorylation profiles of WT, E198K, and E420Kcell lines and find unique and shared changes between variants and WT cells in kinase- and phosphatase-controlled signaling cascades. We observed ribosomal protein S6 (RPS6) hyperphosphorylation as a shared signaling alteration, indicative of increased ribosomal protein S6-kinase activity. Treatment with rapamycin or an RPS6-kinase inhibitor (LY2584702) suppressed RPS6 phosphorylation in both, suggesting upstream activation of mTORC1/p70S6K. Intriguingly, our data suggests ERK-dependent activation of mTORC1 in both E198K and E420K variant cells, with additional AKT-mediated mTORC1 activation in the E420K variant. Thus, although upstream activation of mTORC1 differs between PPP2R5D-related disorder genotypes, inhibition of mTORC1 or RPS6 kinases warrants further investigation as potential therapeutic strategies for patients.

Postexercise High-Fat Feeding Suppresses p70S6K1 Activity in Human Skeletal Muscle.

This study aimed to examine the effects of reduced CHO but high postexercise fat availability on cell signaling and expression of genes with putative roles in regulation of mitochondrial biogenesis, lipid metabolism, and muscle protein synthesis. Ten males completed a twice per day exercise model (3.5 h between sessions) comprising morning high-intensity interval training (8 × 5 min at 85% V˙O2peak) and afternoon steady-state (SS) running (60 min at 70% V˙O2peak). In a repeated-measures design, runners exercised under different isoenergetic dietary conditions consisting of high-CHO (HCHO: 10 g·kg CHO, 2.5 g·kg protein, and 0.8 g·kg fat for the entire trial period) or reduced-CHO but high-fat availability in the postexercise recovery periods (HFAT: 2.5 g·kg CHO, 2.5 g·kg protein, and 3.5 g·kg fat for the entire trial period). Muscle glycogen was lower (P < 0.05) at 3 h (251 vs 301 mmol·kg dry weight) and 15 h (182 vs 312 mmol·kg dry weight) post-SS exercise in HFAT compared with HCHO. Adenosine monophosphate-activated protein kinase α2 activity was not increased post-SS in either condition (P = 0.41), although comparable increases (all P < 0.05) in PGC-1α, p53, citrate synthase, Tfam, peroxisome proliferator-activated receptor, and estrogen-related receptor α mRNA were observed in HCHO and HFAT. By contrast, PDK4 (P = 0.003), CD36 (P = 0.05), and carnitine palmitoyltransferase 1 (P = 0.03) mRNA were greater in HFAT in the recovery period from SS exercise compared with HCHO. Ribosomal protein S6 kinase activity was higher (P = 0.08) at 3 h post-SS exercise in HCHO versus HFAT (72.7 ± 51.9 vs 44.7 ± 27 fmol·min·mg). Postexercise high-fat feeding does not augment the mRNA expression of genes associated with regulatory roles in mitochondrial biogenesis, although it does increase lipid gene expression. However, postexercise ribosomal protein S6 kinase 1 activity is reduced under conditions of high-fat feeding, thus potentially impairing skeletal muscle remodeling processes.

Roles of phosphatidylinositol 3'-kinase and mammalian target of rapamycin/p70 ribosomal protein S6 kinase in K-Ras-mediated transformation of intestinal epithelial cells.

Phosphatidylinositol 3'-kinase (PI3K) activity is required for Ras- mediated transformation of intestinal epithelial cells (IECs). The mammalian target of rapamycin (mTOR) and its downstream pathways control the translation of specific mRNAs that are required for cell proliferation and transformation. Here, we elucidated the roles of PI3K and mTOR in K-Ras-mediated transformation of IECs (IEC-6). Induction of K-Ras activated PI3K and mTOR in IECs. p70 ribosomal protein S6 kinase activity was induced by K-Ras in a PI3K- and mTOR-dependent manner. K-Ras did not significantly alter the phosphorylation of eukaryotic initiation factor 4E-binding protein 1. Treatment with either LY-294002 or rapamycin inhibited IEC proliferation and resulted in G(1) growth arrest. However, it was noted that inhibition of mTOR enhanced K-Ras-mediated morphological transformation and increased invasiveness of IECs in a mitogen-activated protein/extracellular signal-regulated kinase-dependent manner. Furthermore, inhibition of PI3K or mTOR impaired the growth of an array of colon cancer cells. Spindle transformation, reduced E-cadherin, and increased invasiveness were observed in LY-294002-treated Moser cells. Thus, our results suggest that K-Ras-mediated transformation of IECs involves activation of the PI3K/mTOR pathway. Inhibition of PI3K/mTOR activity leads to G(1) growth arrest of transformed IECs. On the other hand, inhibition of PI3K or mTOR may induce the epithelial to mesenchymal transdifferentiation of IECs under certain circumstances.

Sensing the Direction of Stress

Many cells will respond to a tug or a nudge by activating appropriate signaling pathways that ultimately regulate gene expression. But just how cells sense such mechanical strain remains poorly understood. Kumar et al. report observations that begin to reveal previously unrecognized complexity in the sensory machinery. The authors studied responses of diaphragm muscle from mice in vitro. The diaphragm has the unusual property of experiencing mechanical load not only axially, that is, in the direction along the length of the muscle fibers, but also transversely, across the fibers. Both axial and transverse stretching activate the mitogen-activated protein kinases (MAPKs) ERK1 and ERK2, which in turn are linked to activation of p90 RSK (ribosomal protein S6 kinase) and activation of the AP1 transcription factor complex. However, transverse stretching was more effective in activating such signals than was axial stretching. The cell's capacity to detect the direction of mechanical stress was revealed in experiments with pharmacological inhibitors that interfered with specific signaling pathways. Axial stretching activated ERK1 and ERK2 by a mechanism that required signaling through phosphoinositide 3-kinase, intracellular calcium release, protein kinase C, and the MAPK kinases MEK1 or MEK2. In contrast, transverse stretching of the muscle cells activated ERKs 1and 2 in a manner that required cAMP (adenosine 3′-5′ monophosphate)-dependent protein kinase. The authors conclude that diaphragm muscles appear to have two distinct pathways leading to ERK activation and altered gene expression with each responding to mechanical stress in a particular direction. A. Kumar, I. Chaudhry, M. B. Reid, A. M. Boriek, Distinct signaling pathways are activated in response to mechanical stress applied axially and transversely to skeletal muscle fibers. J. Biol. Chem. 277 , 46493-46503 (2002). [Abstract] [Full Text]

Insulin activates glycogen synthase by a novel PI 3-kinase/p70s6k dependent pathway in 3T3-L1 adipocytes.

Insulin causes the activation of a tyrosine kinase activity in the intracellular domain of its receptor, the major substrate of this kinase being the multifunctional docking protein E l . Phosphorylation of specific tyrosine residues on IRSl allows this protein to interact with and activate a number of downstream signalling molecules including phosphoinositide 3-kinase (PI 3-kinase), SHPTP2 and the Grb-2/Sos/ras system (and hence the MAP kinase cascade) 111. Insulin also causes the activation of ribosomal protein S6-kinase (p70dk) by unknown mechanisms [2]. However it is not clear how insulin stimulation of these signalling pathways exerts control over metabolic pathways. We have used rapamyan, which specifically blocks growth factor activation of p70dk, and the specific PI 3kinase inhibitor wortmannin, to investigate the involvement of PI 3-kinase and p706k in insulin stimulation of glucose transport and glycogen synthesis in 3T3-Ll adipocytes. We find that wortmannin inhibits PI 3-kinase activity in anti IRS-1 and anti-PI 3-kinase immunoprecipitates from 3T3-Ll adipocytes in a dose dependent manner (ICm of a 10 nM), with inhibtion being complete at 100 nM (data not shown). Wortmannin inhibited insulin stimulated glucose transport in a similar dose dependent manner in the 3T3-Ll adipocytes (ICm = 15nM) (Fig. 1). The potency of these inhibitory effects of wortmannin agrees with previous reports [3,41. Insulin stimulated glycogen synthase activity 2-3 fold in these cells. Wortmannin inhibited insulin stimulated activation of glycogen synthase with a similar dose dependency to its inhibition of insulin stimulated

Insulin activates a 70-kDa S6 kinase through serine/threonine-specific phosphorylation of the enzyme polypeptide.

The dominant insulin-stimulated ribosomal protein S6 kinase activity was purified to near homogeneity from insulin-treated 32P-labeled rat H4 hepatoma cells and found to copurify with a 70-kDa 32P-labeled polypeptide. The dominant S6 kinase purified from livers of cycloheximide-treated rats is also a 70-kDa polypeptide. Antiserum raised against rat liver S6 kinase specifically immunoprecipitates the purified 32P-labeled H4 hepatoma insulin-stimulated S6 kinase. This antiserum also specifically precipitates insulin-stimulated S6 kinase activity directly from cytosolic extracts of H4 cells. Immune complexes prepared from the cytosol of 32P-labeled H4 cells contain several 32P-labeled polypeptides; only a 70-kDA 32P-labeled peptide, however, is specifically displaced by preadsorption of the antiserum with nonradioactive rat liver S6 kinase. Insulin treatment increases the 32P content of the immunoprecipitated 70-kDa S6 kinase polypeptide 3- to 4-fold over basal levels; 32P-labeled serine, some 32P-labeled threonine, but no 32P-labeled tyrosine are detected after partial acid hydrolysis. Tryptic peptide maps indicate that the insulin-stimulated S6 kinase purified from 32P-labeled H4 cells is phosphorylated at multiple sites distinct from those which participate in autophosphorylation in vitro. Autophosphorylation of rat liver S6 kinase in vitro does not modify S6 kinase activity. The S6 kinases purified from liver of cycloheximide-treated rat and H4 hepatoma insulin-stimulated enzyme are each completely deactivated by incubation with protein phosphatase type 2A in both autophosphorylating and 40S S6 phosphorylating activities. The phosphatase 2A-deactivated 70-kDa S6 kinase is neither reactivated nor phosphorylated by partially purified insulin-stimulated microtubule-associated protein 2 kinase, in experiments where Xenopus S6 kinase II undergoes phosphorylation and partial reactivation. Thus insulin activates the 70-kDa S6 kinase by promoting phosphorylation of specific serine/threonine residues on the enzyme polypeptide, probably through activating an as-yet-unidentified serine/threonine protein kinase distinct from microtubule-associated protein 2 kinase.

In vivo Phosphorylation and Activation of Ribosomal Protein S6 Kinases during Xenopus Oocyte Maturation

Previous studies have shown that increased ribosomal protein S6 kinase activity in unfertilized Xenopus eggs can be resolved by DEAE-Sephacel chromatography into two peaks, designated S6 kinase I and S6 kinase II. We show here that antibody against bacterially expressed S6 kinase II cross-reacts with S6 kinase I. Both S6 kinases undergo marked phosphorylation when they are activated during oocyte maturation, and both become deactivated and dephosphorylated upon activation of eggs. Immunoblotting of extracts of oocytes reveals that all S6 kinase molecules undergo a decrease and increase in electrophoretic mobility upon activation and deactivation, respectively. The increase in electrophoretic mobility can be produced in vitro by incubation of activated S6 kinase with purified phosphatases. Phosphoamino acid analysis of S6 kinase II labeled in vivo during maturation reveals both phosphoserine and phosphothreonine, and phosphopeptide maps suggest that several kinases may phosphorylate and activate S6 kinase II in vivo. These results demonstrate that, during oocyte maturation and early development, S6 kinase activation and deactivation are regulated by phosphorylation and dephosphorylation, suggesting a probable mechanism for S6 kinase regulation in other mitogenically stimulated cells.

Activation of a ribosomal protein S6 kinase in mouse fibroblasts during infection with herpesvirus.

If confluent fibroblasts are infected with the swine alpha-herpes virus, pseudorabies virus, ribosomal protein S6 becomes phosphorylated after a lag of approximately 2 h. When cell-free extracts were prepared from such cells in the presence of glycerol 2-phosphate and EGTA, a ribosomal protein S6 kinase activity was found to appear at approximately the same time as the phosphorylation in vivo. This protein kinase was similar to that activated in the same cells by replenishing the nutrient medium, and in other quiescent cells by the action of growth factors and mitogens. It was distinct from the previously described pseudorabies virus protein kinase, which is unique to infected cells. When medium from cells infected with pseudorabies virus was freed of virus and added to confluent fibroblasts, rapid activation of the ribosomal protein S6 kinase activity occurred. A similar, although more limited, effect could be seen when the pH of the medium was increased. These results suggest that the phosphorylation of ribosomal protein S6 in cells infected with herpes virus is a consequence of the production of a factor which initiates the metabolic programme for cellular growth. The possible function of this effect in the infective strategy of herpes viruses is discussed in relation to requirements for the replication of viral DNA.

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

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, DEAE-cellulose, 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 two-dimensional gel electrophoresis, monophosphorylated and diphosphorylated forms of ribosomal protein S6 were observed to be the predominant radioactively labelled species.

Protein synthesis, ribosomal protein S6 phosphorylation in vitro and the effects of amiloride: SDS gel electrophoresis studies in the Yoshida ascites tumor (AH 130) grown in vivo

Cell-free cytosolic extracts from the Yoshida (AH 130) rat ascites hepatoma cell line, grown in vivo , showed high ribosomal protein S6 kinase activity in vitro , as measured by transfer of 32P to exogenous 40S rat liver ribosomal subunits, in both exponential growing and stationary phase cells. A significant decrease of protein synthesis ( 3H-leucine incorporation into total cell protein) was found to occur in cells reaching the stationary phase of growth, suggesting that S6 phosphorylation was not tightly coupled to the rate of the intraperitoneal cell growth and of protein synthesis in these tumor cells. When the cell-free cytosolic extracts were prepared from cells exposed to amiloride, at concentrations that inhibit the Na + H + exchange, a decrease of S6 kinase activity was observed only in exponential growing cells, suggesting the possibility of coupling of the Na + H + exchange with phosphorylation of intracellular proteins in these tumor cells. Actually, stationary phase cells showed unchanged S6 kinase activity under the same conditions, possibly due to the extremely low Na + H + exchange activity, previously demonstrated (Cell Biol. Int. Rep., 1985, 9, 1017–1025). The present experiments support the hypothesis that the regulation of protein synthesis is not tightly coupled to phosphorylationdephosphorylation cycles, at least of ribosomal protein S6, in cells characterized by a rather uncontrolled growth such as the Yoshida (AH 130) rat ascites hepatoma. In this connection, an elevated degree of protein phosphorylation, such as that of the ribosomal protein S6, could be a general phenomenon of neoplastic transformation.

A similar ribosomal protein S6 kinase activity is found in insulin-treated 3T3-L1 cells and chick embryo fibroblasts transformed by Rous sarcoma virus

Insulin and transformation by Rous sarcoma virus stimulate the phosphorylation of ribosomal protein S6. Soluble fractions containing activated S6 protein kinase from insulin-treated cells and from transformed chick embryo fibroblasts were compared. Based upon several characteristics notably elution from DEAE-cellulose and sedimentation in glycerol gradients, these two S6 protein kinase activities appear to be similar enzymes. Thus insulin and retroviral transformation may activate the same enzyme to regulate the phosphorylation state of S6.

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.

Regulation of a ribosomal protein S6 kinase activity by the Rous sarcoma virus transforming protein, serum, or phorbol ester.

Protein kinase capable of phosphorylating 40S ribosomal protein S6 on serine residues has been detected in chicken embryo fibroblasts. This activity appears to be regulated in direct response to expression of pp60v-src in chicken embryo fibroblasts infected with a temperature-sensitive transformation mutant of Rous sarcoma virus. Partially purified S6 kinase was highly specific for S6 in 40S ribosomal subunits. The S6 kinase was not inhibited by calcium or by the heat-stable inhibitor of cAMP-dependent protein kinase, nor was it activated by phosphatidylserine, diacylglycerol, and calcium. Thus, it is distinct from protein kinase C and cAMP-dependent protein kinase, which are capable of phosphorylating S6 in vitro. The tumor-promoter phorbol 12-myristate 13-acetate also stimulated ribosomal protein S6 kinase activity in serum-starved chicken embryo fibroblasts, whereas phorbol, the inactive analog of phorbol 12-myristate 13-acetate, had no effect. S6 kinase activity stimulated by expression of pp60v-src, by phorbol 12-myristate 13-acetate, or by serum growth factors exhibited similar chromatographic properties upon ion-exchange chromatography. These results suggest that a common protein kinase may be activated by three diverse stimuli all involved in regulating cell proliferation.