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(beta-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.     

Protein phosphorylation and protein kinase activities in BC3H-1 myocytes. Differences between the effects of insulin and phorbol esters

To determine whether insulin activates protein kinase C in BC3H-1 myocytes, we evaluated changes in protein phosphorylation, protein kinase activities, and the intracellular translocation of protein kinase C activity in response to insulin and phorbol esters. Phorbol 12-myristate 13-acetate (PMA), but not insulin, stimulated the phosphorylation of an acidic Mr 80,000 protein which has been shown to be an apparently specific marker for protein kinase C activation. In addition, PMA, but not insulin, stimulated the rapid association of protein kinase C activity with a cellular particulate fraction. In contrast to these differences, both insulin and PMA stimulated the phosphorylation of ribosomal protein S6 and activated a ribosomal protein S6 kinase in cell-free extracts from cells exposed to these agents. In cells exposed to high concentrations of PMA for 16 h, protein kinase C activity and immunoreactivity were abolished, without changes in cellular morphology. Under these conditions, insulin, but not PMA, stimulated phosphorylation of the ribosomal protein S6 in intact cells and activated the S6 kinase in cell-free extracts derived from insulin-treated intact cells. We conclude that: insulin does not appear to activate protein kinase C in BC3H-1 myocytes, at least as assessed by phosphorylation of the Mr 80,000 protein; both insulin and PMA activate an S6 protein kinase in these cells; and insulin can promote S6 phosphorylation and activate the S6 kinase normally in protein kinase C-deficient cells. Activation of the S6 kinase by insulin and PMA, although apparently proceeding through different mechanisms, may explain some of the similar biological actions of these compounds in BC3H-1 myocytes.     

An activated S6 kinase in extracts from serum- and epidermal growth factor-stimulated Swiss 3T3 cells

Soluble extracts from serum- or epidermal growth factor-stimulated Swiss 3T3 cells show up to a 25-fold increase in their ability to phosphorylate 40 S ribosomal protein S6. The increased S6 phosphorylation is due to increased protein kinase activity in extracts of stimulated cells and not due to the inactivation of an S6 phosphatase. However, the presence of phosphatase inhibitors as well as EGTA is required during the preparation of cell extracts to obtain fully active S6 kinase(s). Epidermal growth factor has little effect at concentrations below 10(-10) M: activity increases sharply at 10(-9) M epidermal growth factor and reaches saturation at 10(-8) M (50-60% of the activity obtained by stimulating with 10% serum). Activation of the kinase activity in cell extracts is observed as early as 2 min after serum stimulation, reaches 50% between 10 and 15 min, and is maximal by 60 min of serum stimulation. Phosphorylation in vitro of ribosomal protein S6 with extracts from serum-stimulated cells followed by analysis of the tryptic phosphopeptides shows the presence of 9 of the 11 phosphopeptides induced by serum in vivo.     

Glucosamine-induced insulin resistance in 3T3-L1 adipocytes

To study molecular mechanisms for glucosamine-induced insulin resistance, we induced complete and reversible insulin resistance in 3T3-L1 adipocytes with glucosamine in a dose- and time-dependent manner (maximal effects at 50 mM glucosamine after 6 h). In these cells, glucosamine impaired insulin-stimulated GLUT-4 translocation. Glucosamine (6 h) did not affect insulin-stimulated tyrosine phosphorylation of the insulin receptor and insulin receptor substrate-1 and -2 and weakly, if at all, impaired insulin stimulation of phosphatidylinositol 3-kinase. Glucosamine, however, severely impaired insulin stimulation of Akt. Inhibition of insulin-stimulated glucose transport was correlated with that of Akt activity. In these cells, glucosamine also inhibited insulin stimulation of p70 S6 kinase. Glucosamine did not alter basal glucose transport and insulin stimulation of GLUT-1 translocation and mitogen-activated protein kinase. In summary, glucosamine induced complete and reversible insulin resistance in 3T3-L1 adipocytes. This insulin resistance was accompanied by impaired insulin stimulation of GLUT-4 translocation and Akt activity, without significant impairment of upstream molecules in insulin-signaling pathway.     

Defective regulation of mitogen-activated protein kinase activity in a 3T3 cell variant mitogenically nonresponsive to tetradecanoyl phorbol acetate.

Mitogen-activated protein (MAP) kinase is a serine/threonine-specific protein kinase which is activated in response to various mitogenic agonists (e.g., epidermal growth factor, insulin, and the tumor promoter tetradecanoyl phorbol acetate [TPA]) and requires both threonine and tyrosine phosphorylation for activity. This enzyme has recently been shown to be identical or closely related to pp42, a protein which becomes tyrosine phosphorylated in response to mitogenic stimulation. Neither the kinases which regulate MAP kinase/pp42 nor the in vivo substrates for this enzyme are known. Because MAP MAP kinase is activated and phosphorylated in response both to agents which stimulate tyrosine kinase receptors and to agents which stimulate protein kinase C, a serine/threonine kinase, we have examined the regulation and phosphorylation of this enzyme in 3T3-TNR9 cells, a variant cell line partially defective in protein kinase C-mediated signalling. In this communication, we show that in the 3T3-TNR9 variant cell line, TPA does not cause the characteristically rapid phosphorylation of pp42 or the activation and phosphorylation of MAP kinase. This defective response is not due to the absence of the MAP kinase/pp42 protein itself because both tyrosine phosphorylation of MAP kinase/pp42 and its enzymatic activation could be induced by platelet-derived growth factor in the 3T3-TNR9 cells. Thus, the defect in these variant cells apparently resides in some aspect of the regulation of MAP kinase phosphorylation. Since the 3T3-TNR9 cells are also defective with respect to the TPA-induced increase in ribosomal protein S6 kinase, these in vivo results reinforce the earlier in vitro finding that MAP kinase can regulate S6 kinase activity. These findings suggest a key role for MAP kinase in a kinase cascade cascade involved in the control of cell proliferation.     

Monoclonal antibodies to the human insulin receptor mimic a spectrum of biological effects in transfected 3T3/HIR fibroblasts without activating receptor kinase

The effects of four monoclonal antibodies to the alpha subunit of the human insulin receptor were studied in transfected mouse 3T3 fibroblasts expressing human insulin receptors (3T3/HIR). Three antibodies, MA-5, MA-20, and MA-51, mimicked insulin stimulation of the uptake of both 2-deoxy-D-glucose and alpha-aminoisobutyrio acid, and S6 kinase activity. Antibody MA-5 also mimicked insulin stimulation of [3H]thymidine incorporation and cell growth. Although these antibodies mimicked insulin stimulation of biological effects, they failed to significantly activate insulin receptor tyrosine kinase activity. These studies suggest, therefore, that the insulin receptor can signal a variety of cellular functions without stimulation of receptor kinase activity.     

Monoclonal antibodies mimic insulin activation of ribosomal protein S6 kinase without activation of insulin receptor tyrosine kinase. Studies in cells transfected with normal and mutant human insulin receptors

The effects of species-specific monoclonal antibodies to the human insulin receptor on ribosomal protein S6 phosphorylation were studied in rodent cell lines transfected with human insulin receptors. First, Swiss mouse 3T3 fibroblasts expressing normal human insulin receptors (3T3/HIR cells) were studied. Three monoclonal antibodies, MA-5, MA-20, and MA-51, activated S6 kinase in these cells but had no effects in untransfected 3T3 cells. Both insulin and MA-5, the most potent antibody, activated S6 kinase in a similar time- and dose-dependent manner. To measure S6 phosphorylation in vivo, 3T3/HIR cells were preincubated with [32P]Pi and treated with insulin and MA-5. Both agents increased S6 phosphorylation, and their tryptic phosphopeptide maps were similar. MA-5 and the other monoclonal antibodies, unlike insulin, failed to stimulate insulin receptor tyrosine kinase activity either in vitro or in vivo. Moreover, unlike insulin, they failed to increase the tyrosine phosphorylation of the endogenous cytoplasmic protein, pp 185. Next, HTC rat hepatoma cells, expressing a human insulin receptor mutant that had three key tyrosine autophosphorylation sites in the beta-subunit changed to phenylalanines (HTC-IR-F3 cells), were studied. In this cell line but not in untransfected HTC cells, monoclonal antibodies activated S6 kinase without stimulating either insulin receptor autophosphorylation or the tyrosine phosphorylation of pp 185. These data indicate, therefore, that monoclonal antibodies can activate S6 kinase and then increase S6 phosphorylation. Moreover, they suggest that activation of receptor tyrosine kinase and subsequent tyrosine phosphorylation of cellular proteins may not be crucial for activation of S6 kinase by the insulin receptor.     

Differential stimulation of phosphorylation of initiation factors eIF-4F, eIF-4B, eIF-3, and ribosomal protein S6 by insulin and phorbol esters

Exposure of quiescent, serum-starved 3T3-L1 cells to insulin promotes phosphorylation of initiation factors eIF-4F, eIF-4B, and eIF-3 p120, as well as ribosomal protein S6. Phosphorylation of both the p25 and p220 subunits of eIF-4F is stimulated typically by 2.5-5-fold, with a 2-4-fold increase in phosphorylation of eIF-4B and eIF-3 p120. Optimal stimulation is observed by 10(-9) M insulin. A similar pattern of stimulation is seen upon treatment of 3T3-L1 cells with 1 x 10(-6) M phorbol 12-myristate 13-acetate (PMA). Two-dimensional phosphopeptide mapping of p25, isolated from quiescent, insulin- or PMA-stimulated cells, results in a single tryptic phosphopeptide, indicating a single phosphorylation site identical to that obtained with protein kinase C. A more complex phosphopeptide map is observed with the p220 subunit. Following PMA-stimulation of 3T3-L1 cells, phosphopeptide mapping of p220 results in a pattern similar to that observed in vitro with Ca2+/phospholipid-dependent protein kinase (protein kinase C). Following insulin stimulation, mapping of p220 results in the appearance of novel peptides. Upon prolonged exposure to PMA, the cells are no longer responsive to this mitogen and no stimulation of phosphorylation of eIF-4F, eIF-4b, eIF-3 p120, or S6 via a protein kinase C-dependent mechanism is observed. Addition of insulin to these down-regulated cells leads to stimulation of phosphorylation of eIF-4F p220, ribosomal protein S6, and to a lesser extent, eIF-4B; little or no stimulation of phosphorylation of eIF-4F p25 and eIF-3 p120 is observed. Thus, eIF-4F p220, eIF-4B and ribosomal protein S6 are phosphorylated via PMA-dependent and insulin-dependent pathways, whereas phosphorylation of eIF-4F p25 and eIF-3 p120 is stimulated only upon activation of protein kinase C. Phosphopeptide maps of eIF-4F p220 and ribosomal protein S6 suggest that protease-activated kinase II is one of the protein kinases involved in the insulin-stimulated response in protein kinase C-depleted cells.     

Protease-activated kinase II as the potential mediator of insulin-stimulated phosphorylation of ribosomal protein S6

One of the earliest responses to insulin in target cells is stimulation of the phosphorylation of ribosomal protein S6. When exponentially growing 3T3-L1 cells are serum-starved, little phosphorylation of S6 is observed; however, following addition of insulin (10(-7) M), up to 5 phosphoryl groups are incorporated into S6. An enzyme mediating the insulin-stimulated phosphorylation of S6 has been identified as protease-activated kinase II. Two-dimensional peptide maps of tryptic digests of S6 from insulin-treated 3T3-L1 cells contain 5 phosphopeptides; the same 5 phosphopeptides are observed with tryptic digests of 40 S ribosomal subunits phosphorylated in vitro by protease-activated kinase II from rabbit reticulocytes. Protease-activated kinase II has also been identified and partially purified from the postribosomal supernatant of serum-starved and insulin-treated 3T3-L1 cells. The enzyme is present in the inactive proenzyme form in serum-starved cells; following insulin treatment, approximately 50% of the enzyme is in an activated form. Identical tryptic phosphopeptide maps are observed with these enzymes.     

Amino acid and insulin signaling via the mTOR/p70 S6 kinase pathway. A negative feedback mechanism leading to insulin resistance in skeletal muscle cells

Amino acids have emerged as potent modulators of the mTOR/p70 S6 kinase pathway. The involvement of this pathway in the regulation of insulin-stimulated glucose transport was investigated in the present study. Acute exposure (1 h) to a balanced mixture of amino acids reduced insulin-stimulated glucose transport by as much as 55% in L6 muscle cells. The effect of amino acids was fully prevented by the specific mTOR inhibitor rapamycin. Time course analysis of insulin receptor substrate 1 (IRS-1)-associated phosphatidylinositol (PI) 3-kinase activity revealed that incubation with amino acids speeds up its time-dependent deactivation, leading to a dramatic suppression (-70%) of its activity after 30 min of insulin stimulation as compared with its maximal activation (5 min of stimulation). This accelerated deactivation of PI 3-kinase activity in amino acid-treated cells was associated with a concomitant and sustained increase in the phosphorylation of p70 S6 kinase. In marked contrast, inhibition of mTOR by rapamycin maintained PI 3-kinase maximally activated for up to 30 min. The marked inhibition of insulin-mediated PI 3-kinase activity by amino acids was linked to a rapamycin-sensitive increase in serine/threonine phosphorylation of IRS-1 and a decreased binding of the p85 subunit of PI 3-kinase to IRS-1. Furthermore, amino acids were required for the degradation of IRS-1 during long term insulin treatment. These results identify the mTOR/p70 S6 kinase signaling pathway as a novel modulator of insulin-stimulated glucose transport in skeletal muscle cells.     

Regulation of the insulin receptor kinase by hyperinsulinism

A murine fibroblast cell line transfected with human insulin receptor cDNA, NIH 3T3 HIR3.5, was observed to display insulin-induced down-regulation of insulin-binding activity in a time- and concentration-dependent manner. Maximal inhibition of insulin-binding activity (54%) occurred within 16 h of exposure to 100 nM insulin in vivo, where in vivo refers to intact cells in tissue culture. The decrease in cellular insulin-binding activity was the consequence of a decrease in the number of cell-associated insulin receptors as determined by Scatchard analysis of insulin binding, 125I-insulin affinity cross-linking, and Western blotting of the insulin receptor beta subunit. Acute insulin treatment in vivo (1-60 min) resulted in the activation of the insulin receptor protein tyrosine kinase as determined by in vitro phosphorylation of glutamic acid:tyrosine (4:1), where in vitro refers to broken cell preparations. This acute in vivo insulin activation of the insulin receptor tyrosine kinase resulted in a greater stimulation (1.4-1.9-fold) of tyrosine kinase activity in the glutamic acid:tyrosine (4:1) assay than the maximal stimulation produced by insulin treatment in vitro. In contrast, long term (24 h) insulin treatment in vivo resulted in a 50-70% decrease in intrinsic protein tyrosine kinase activity of the insulin receptors compared with that of acutely activated (1 min) insulin receptors. Under these conditions, the insulin receptor protein kinase activity remained insulin independent in the in vitro substrate kinase assay. Surprisingly, the insulin-independent activated (1 min in vivo insulin-treated) and uncoupled (24 h in vivo insulin-treated) insulin receptors displayed similar stoichiometries of 32P incorporation into the beta subunit by in vitro autophosphorylation when compared with the control insulin receptors, ranging from 1.5 to 1.8 mol of phosphate incorporated/mol of insulin receptor. Phosphoamino acid analysis demonstrated that the phosphoserine/phosphothreonine content of in vivo 32P-labeled insulin receptors increased markedly within a 1-h exposure to insulin in vivo, whereas insulin-induced receptor desensitization was not apparent until 10-24 h after exposure to insulin. These data suggest that insulin treatment in vivo results initially in the activation of the insulin receptor kinase followed by a subsequent uncoupling of protein kinase activity. This insulin-induced desensitization of the insulin receptor kinase does not correlate with the extent of beta subunit serine/threonine phosphorylation.     

Protein phosphatase 2A inactivates the mitogen-stimulated S6 kinase from Swiss mouse 3T3 cells

Treatment of quiescent 3T3 cells with sodium orthovanadate induces a 10-fold stimulation of a kinase that phosphorylates ribosomal protein S6. The kinase in crude extracts is extremely labile and rapidly loses activity when incubated at 37 degrees C. This reaction is blocked by phosphatase inhibitors such as p-nitrophenyl phosphate and beta-glycerophosphate, suggesting that dephosphorylation of the kinase leads to its inactivation (Novak-Hofer, I., and Thomas, G. (1985) J. Biol. Chem. 260, 10314-10319). After three steps of purification the kinase can be separated from greater than 99% of the cellular phosphorylase a phosphatases. At this stage the kinase preparation is almost completely stable but can be inactivated by readdition of specific column fractions that contain both phosphorylase phosphatase and protease activity. However, employing a number of specific inhibitors it is shown that the inactivating agent in these fractions is a protein phosphatase. Furthermore, the physical and enzymatic properties of the kinase inactivator argue that it can be classified as a type 2A phosphatase. These results are consistent with the finding that the purified catalytic subunits of phosphatase type 1 and type 2A also inactivate the kinase. At equivalent phosphorylase a phosphatase activities, the type 2A catalytic subunit is 3 times more potent than the type 1 enzyme in carrying out this reaction. These data indicate that the major S6 kinase inactivator in 3T3 cell extracts is a type 2A phosphatase, supporting the hypothesis that the orthovanadate-stimulated S6 kinase is regulated in vivo by a phosphorylation-dephosphorylation mechanism.     

Mitogen-activated S6 kinase is stimulated via protein kinase C-dependent and independent pathways in Swiss 3T3 cells

Soluble extracts prepared from quiescent Swiss mouse 3T3 cells that had been briefly exposed to various mitogens exhibited a 2- to 3-fold elevation in phosphorylating activities toward ribosomal protein S6 and a synthetic peptide, Arg-Arg-Leu-Ser-Ser-Leu-Arg-Ala (RRLSSLRA), patterned after a phosphorylation site sequence from S6. Optimal activation of the phosphorylating activity occurred within 15-20 min of exposure of the cells to platelet-derived growth factor (10 ng/ml), epidermal growth factor (100 nM), and insulin (100 nM), and 2-5 min after 12-O-tetradecanoylphorbol-13-acetate (TPA) (100 nM) treatment. Fractionation of the cytosolic extracts from mitogen- or TPA-treated cells on Sephacryl S-300, TSK-400, and DEAE-Sephacel columns gave results suggesting that a single stimulated kinase accounted for the enhanced S6 and RRLSSLRA phosphorylating activities. The mitogen-activated kinase had an apparent Mr of about 85,000 as determined with Sephacryl S-300, but eluted with an apparent Mr of 26,000 from a TSK-400 high pressure liquid chromatography column. The S6 kinase was also stimulated in cytosols from insulin-like growth factor 1- (100 nM), vasopressin- (250 nM), prostaglandin F2 alpha- (250 nM), and 10% fetal calf serum-treated cells but not from quiescent cells exposed to beta-transforming growth factor (2 ng/ml). TPA, vasopressin and prostaglandin F2 alpha appeared to stimulate this kinase via a protein kinase C-dependent mechanism, since the responses to these hormones, but not to platelet-derived growth factor, epidermal growth factor, and insulin, were lost in protein kinase C-depleted cells.     

Restraining PI3K: mTOR signalling goes back to the membrane

The lipid kinase phosphoinositide 3-kinase (PI3K) is activated in response to various extracellular signals including peptide growth factors such as insulin and insulin-like growth factors (IGFs). Phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P(3)] generated by PI3K is central to the diverse responses elicited by insulin, including glucose homeostasis, proliferation, survival and cell growth. The actions of lipid phosphatases have been considered to be the main means of attenuating PI3K signalling, whereby the principal 3-phosphatase - phosphatase and tensin homologue deleted on chromosome 10 (PTEN) - dephosphorylates PtdIns(3,4,5)P(3), reversing the action of PI3K. Recently, however, another pathway of regulation of PI3K has been identified in which activation of PI3K itself is prevented. This finding, together with earlier work, strongly suggests that a major form of negative feedback inhibition of PI3K results from activated growth signalling via mammalian target of rapamycin (mTOR) and the p70 S6 kinase (S6K) - a pathway that could have consequences for the development of type 2 diabetes and tuberous sclerosis complex.     

The TSC1-2 tumor suppressor controls insulin-PI3K signaling via regulation of IRS proteins

Insulin-like growth factors elicit many responses through activation of phosphoinositide 3-OH kinase (PI3K). The tuberous sclerosis complex (TSC1-2) suppresses cell growth by negatively regulating a protein kinase, p70S6K (S6K1), which generally requires PI3K signals for its activation. Here, we show that TSC1-2 is required for insulin signaling to PI3K. TSC1-2 maintains insulin signaling to PI3K by restraining the activity of S6K, which when activated inactivates insulin receptor substrate (IRS) function, via repression of IRS-1 gene expression and via direct phosphorylation of IRS-1. Our results argue that the low malignant potential of tumors arising from TSC1-2 dysfunction may be explained by the failure of TSC mutant cells to activate PI3K and its downstream effectors.     

Activation of a Ca2+-inhibitable protein kinase that phosphorylates microtubule-associated protein 2 in vitro by growth factors, phorbol esters, and serum in quiescent cultured human fibroblasts

Treatment of quiescent human embryonic lung fibroblastic cells (TIG-3) with 10 nM epidermal growth factor (EGF) resulted in 4-6-fold activation of a protein kinase activity in cell extracts that phosphorylated microtubule-associated protein 2 (MAP2) on serine and threonine residues in vitro. The half-maximal activation of the kinase activity occurred within 5 min after EGF treatment, and the maximal level was attained at 15 min. Casein and histone were very poor substrates for this EGF-stimulated MAP2 kinase activity. The activation of the kinase activity persisted after brief dialysis. Interestingly, the EGF-stimulated MAP2 kinase activity was sensitive to micromolar concentrations of free Ca2+; it was inhibited 50% by 0.5 microM Ca2+ and almost totally inhibited by 2 microM Ca2+. The activated MAP2 kinase activity was recovered in flow-through fractions on phosphocellulose column chromatography, while kinase activities that phosphorylate 40 S ribosomal protein S6 (S6 kinase activities) were mostly retained on the column and eluted at 0.5 M NaCl. Platelet-derived growth factor, fibroblast growth factor, insulin-like growth factor-I, insulin, phorbol esters (12-O-tetradecanoylphorbol 13-acetate and phorbol 12,13-dibutyrate), and fresh fetal calf serum also induced activation of the MAP2 kinase in the quiescent TIG-3 cells. The activated MAP2 kinase activity in cells stimulated by platelet-derived growth factor, fibroblast growth factor, insulin-like growth factor-I, insulin, 12-O-tetradecanoylphorbol 13-acetate, phorbol 12,13-dibutyrate, or fetal calf serum was almost completely inhibited by 2 microM Ca2+, like the EGF-stimulated kinase. In addition, MAP2 phosphorylated by the kinase activated by different stimuli gave very similar phosphopeptide mapping patterns. These results suggest that several growth factors, phorbol esters, and serum activate a common, Ca2+-inhibitable protein kinase which is distinct from S6 kinase in quiescent human fibroblasts.     

Stimulation of protein synthesis, eukaryotic translation initiation factor 4E phosphorylation, and PHAS-I phosphorylation by insulin requires insulin receptor substrate 1 and phosphatidylinositol 3-kinase.

Insulin rapidly stimulates protein synthesis in a wide variety of tissues. This stimulation is associated with phosphorylation of several translational initiation and elongation factors, but little is known about the signaling pathways to these events. To study these pathways, we have used a myeloid progenitor cell line (32D) which is dependent on interleukin 3 but insensitive to insulin because of the very low levels of insulin receptor (IR) and the complete lack of insulin receptor substrate (IRS)-signaling proteins (IRS-1 and IRS-2). Expression of more IR permits partial stimulation of mitogen-activated protein kinase by insulin, and expression of IRS-1 alone mediates insulin stimulation of the 70-kDa S6 kinase (pp70S6K) by the endogenous IR. However, expression of both IR and IRS-1 is required for stimulation of protein synthesis. Moreover, this effect requires activation of phosphatidylinositol 3-kinase (PI3K), as determined by wortmannin inhibition and the use of an IRS-1 variant lacking all Tyr residues except those which activate PI3K. Stimulation of general protein synthesis does not involve activation by IRS-1 of GRB-2-SOS-p21ras or SH-PTP2, since IRS-1 variants lacking the SH2-binding Tyr residues for these proteins are fully active. Nor does it involve pp70S6K, since rapamycin, while strongly inhibiting the synthesis of a small subset of growth-regulated proteins, only slightly inhibits total protein synthesis. Recruitment of mRNAs to the ribosome is enhanced by phosphorylation of eIF4E, the cap-binding protein, and PHAS-I, a protein that specifically binds eIF4E. The behavior of cell lines containing IRS-1 variants and inhibition by wortmannin and rapamycin indicate that the phosphorylation of both proteins requires IRS-1-mediated stimulation of PI3K and pp70S6K but not mitogen-activated protein kinase or SH-PTP2.     

Distinct signalling pathways mediate insulin and phorbol ester-stimulated eukaryotic initiation factor 4F assembly and protein synthesis in HEK 293 cells

Stimulation of serum-starved human embryonic kidney (HEK) 293 cells with either the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), or insulin resulted in increases in the phosphorylation of 4E-BP1 and p70 S6 kinase, eIF4F assembly, and protein synthesis. All these effects were blocked by rapamycin, a specific inhibitor of mTOR. Phosphatidylinositol 3-kinase and protein kinase B were activated by insulin but not by TPA. Therefore TPA can induce eIF4F assembly, protein synthesis, and the phosphorylation of p70 S6 kinase and 4E-BP1 independently of both phosphatidylinositol 3-kinase and protein kinase B. Using two structurally unrelated inhibitors of MEK (PD098059 and U0126), we provide evidence that Erk activation is important in TPA stimulation of eIF4F assembly and the phosphorylation of p70 S6 kinase and 4E-BP1 and that basal MEK activity is important for basal, insulin, and TPA-stimulated protein synthesis. Transient transfection of constitutively active mitogen-activated protein kinase interacting kinase 1 (the eIF4E kinase) indicated that inhibition of protein synthesis and eIF4F assembly by PD098059 is not through inhibition of eIF4E phosphorylation but of other signals emanating from MEK. This report also provides evidence that increased eIF4E phosphorylation alone does not affect the assembly of the eIF4F complex or general protein synthesis.     

Identification of multiple epidermal growth factor-stimulated protein serine/threonine kinases from Swiss 3T3 cells

Growth factor activation of serine/threonine protein kinases was studied by treating quiescent Swiss 3T3 cells with epidermal growth factor (EGF) and examining cytosolic extracts for protein kinase activity under conditions inhibitory to calcium- and cyclic nucleotide-dependent kinases. Cytosolic extracts of cells stimulated for 5 min were fractionated by Mono Q fast protein liquid chromatography. Eight peaks of kinase activity were resolved, of which five were stimulated by EGF treatment of cells. These peaks were revealed using the synthetic peptide Arg-Arg-Leu-Ser-Ser-Leu-Arg-Ala (S6 peptide), 40 S ribosomal S6 protein, glycogen synthase, microtubule-associated protein 2, and myelin basic protein as substrates. The peaks varied in the kinetics of their activation by EGF and in their response to insulin. Selected peaks were resolved further by sizing gel chromatography. The results together indicate that at least seven distinct fractions of cytosolic kinase activities are stimulated in Swiss 3T3 cells by EGF. One of these, which phosphorylates both S6 protein and S6 peptide, is similar to the S6 kinase characterized previously in this cell line by others. Four additional activities that also phosphorylate the S6 protein and S6 peptide appear unrelated to this enzyme. Finally, two kinase activities that phosphorylate both myelin basic protein and microtubule associated protein 2 are EGF stimulated. One is similar to an insulin-stimulated microtubule-associated protein 2 kinase described in other cell lines whereas the other seems to represent a novel activity. Several of these EGF-stimulated activities were inactivated by protein phosphatases, suggesting that they might be regulated by phosphorylation.