Purification of a hepatic S6 kinase from cycloheximide-treated Rats

Cycloheximide injection of rats results in the activation of a protein kinase that phosphorylates 40 S ribosomal protein S6. This Ca2+/cyclic nucleotide-independent kinase exhibits chromatographic properties that are indistinguishable from the S6 kinase in H4 hepatoma cells whose activity is stimulated by insulin and growth factors and the S6 kinase that is activated during liver regeneration. The enzyme has been purified 50,000-fold to near homogeneity: a critical step in purification employs a peptide affinity column using a synthetic peptide corresponding to the carboxyl-terminal 32-amino acid residues of mouse liver S6, which encompasses all S6 phosphorylation sites. The purified enzyme is a 70,000-dalton polypeptide that is reactive with azido-ATP. In addition to 40 S ribosomal S6 and the synthetic peptide, the S6 kinase catalyzes rapid phosphorylation of a number of other protein substrates including histone H2b, glycogen synthase, and ATP citrate lyase; this last protein is phosphorylated by S6 kinase in vitro on the same serine residue that is phosphorylated in response to insulin and epidermal growth factor in intact hepatocytes. Moreover, the S6 kinase catalyzes the phosphorylation of a number of hepatic nonhistone nuclear proteins. This S6 kinase probably underlies the increased hepatic S6 phosphorylation observed after cycloheximide treatment, which in turn corresponds to the mitogen-activated S6 kinase.     

Regulation of 40S ribosomal protein S6 phosphorylation in Swiss mouse 3T3 cells

Addition of serum to resting cultures of Swiss mouse 3T3 cells causes an immediate multiple phosphorylation of 40S ribosomal protein S6. After 60 min of stimulation, changing to medium containing no serum led to the net dephosphorylation of S6. During this same period, a second protein, as yet unidentified, became increasingly phosphorylated. Incubation of cells with cycloheximide prior to the addition of serum almost completely blocked the activation of protein synthesis. There was no effect on the serum-induced phosphorylation of S6. If cells were stimulated in the presence of cAMP phosphodiesterase inhibitors theophylline or SQ 20006, both S6 phosphorylation and the activation of protein synthesis were inhibited. Stimulation of cells with serum also led to an immediate drop in total intracellular cAMP levels. This was blocked by prostaglandin E1 (PGE1), which caused a 10 fold increase in total intracellular cyclic AMP. However, PGE1 had no effect on protein synthesis or S6 phosphorylation.     

Post-transcriptional regulation by insulin of Xenopus ribosomal protein S6 kinase

The activation of Xenopus oocyte ribosomal protein S6 kinase during oocyte maturation was investigated. Insulin treatment caused a rapid three-fold activation of S6 kinase that returned to near basal levels by 2 h postinsulin. This was followed by a later fivefold increase from 2 to 5 h with insulin, culminating with germinal vesicle breakdown. Pretreatment of oocytes with multiple protein synthesis inhibitors increased the level of basal activity, but did not greatly alter the time course of early activation of S6 kinase by insulin. In contrast, the later increase in S6 kinase activity was completely inhibited by pretreatment with cycloheximide. However, near maximal increases in S6 kinase activity occurred following injection of maturation-promoting factor, even in the presence of multiple protein synthesis inhibitors. Brief exposure to cycloheximide after 30 min or more of insulin stimulation increased the magnitude of insulin-stimulated activity without changing the overall pattern of activity increase. These results suggest that a rapidly turning-over inhibitor of S6 kinase exists, and the activation of S6 kinase by insulin occurs by protein synthesis-dependent and -independent mechanisms.     

Role of protein kinase C in arginine vasopressin-stimulated ERK and p70S6 kinase phosphorylation

We previously showed in rat renal glomerular mesangial cells, that arginine vasopressin (AVP)-stimulated cell proliferation was mediated by epidermal growth factor receptor (EGF-R) transactivation, and activation (phosphorylation) of ERK1/2 and p70S6 kinase (Ghosh et al. [2001]: Am J Physiol Renal Physiol 280:F972-F979]. In this paper, we extend these observations and show that different protein kinase C (PKC) isoforms play different roles in mediating AVP-stimulated ERK1/2 and p70S6 kinase phosphorylation and cell proliferation. AVP treatment for 0-60 min stimulated the serine/threonine phosphorylation of PKC isoforms alpha, delta, epsilon, and zeta. The activation of PKC was dependent on EGF-R and phosphatidylinositol 3-kinase (PI3K) activation. In addition, inhibition of conventional and novel PKC isoforms by chronic (24 h) exposure to phorbol 12-myristate 13-acetate (PMA) inhibited AVP-induced activation of ERK and p70S6 kinase as well as EGF-R phosphorylation. Rottlerin, a specific inhibitor of PKCdelta, inhibited both ERK and p70S6 kinase phosphorylation and cell proliferation. In contrast, a PKCepsilon translocation inhibitor decreased ERK1/2 activation without affecting p70S6 kinase or cell proliferation, while a dominant negative PKCzeta (K281W) cDNA delayed p70S6 kinase activation without affecting ERK1/2. On the other hand, G?6976, an inhibitor of conventional PKC isoforms, did not affect p70S6 kinase, but stimulated ERK1/2 phosphorylation without affecting cell proliferation. Our results indicate that PKCdelta plays an important role in AVP-stimulated ERK and p70S6 kinase activation and cell proliferation.     

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.     

Phosphorylation of ribosomal protein S6 at multiple sites by a cyclic AMP-independent protein kinase from lymphoid cells

Ribosomes prepared from murine lymphosarcoma cells were phosphorylated by a cyclic AMP-independent protein kinase designated H4P kinase. H4P kinase was isolated as an inactive enzyme which was activated by Mg2+-ATP and an endogenous converting enzyme. In the absence of preactivation by Mg2+-ATP and an endogenous converting enzyme, H4P kinase catalyzed phosphorylation of 80, 60, and 40 S ribosomal subunits at a low rate. After activation, the H4P kinase selectively catalyzed phosphorylation of the S 6 protein in the 40 S ribosomal subunit. Under the assay conditions selected, at least 90% of the [32P]phosphate transferred to the 40 S ribosomal preparation was incorporated into S 6. The apparent Km for 40 S subunits phosphorylated by H4P kinase was 7.2 microM. The calculated Vmax was 50 nmol of Pi transferred per min/mg. Exhaustive phosphorylation of 40 S subunits resulted in incorporation of 3 mol of phosphate/mol of S 6, in contrast to results reported previously which indicated 0.3 mol of phosphate was transferred by a similar enzyme from reticulocyte (Del Grande, R. W., and Traugh, J. A. (1982) Eur. J. Biochem. 123, 421-428). These data are consistent with a potential role for H4P kinase in the insulin-mediated phosphorylation of S 6 at multiple sites.     

An insulin-stimulated ribosomal protein S6 kinase in 3T3-L1 cells

A protein kinase that is stimulated from 2-10-fold by insulin and that phosphorylates ribosomal protein S6 has been characterized in 3T3-L1 cells. The detection of this activity in the 100,000 X g supernatant is facilitated by the presence of beta-glycerol phosphate or vanadate in the homogenization buffer. The activity has been purified 55-fold by chromatography on DEAE-cellulose and phosphocellulose. The resulting specific activity is 584 pmol/min/mg of protein. DEAE-cellulose chromatography followed by gel filtration on Ultrogel AcA54 or by glycerol gradient centrifugation suggests that the protein has a molecular mass of 60,000-70,000 daltons. Mg2+, and to a lesser extent Mn2+, will support phosphorylation of S6 by the activity. No proteins tested other than ribosomal protein S6 are phosphorylated. Based on its chromatographic properties and substrate specificity, the enzyme appears to be distinct from several other protein kinases that are known to phosphorylate ribosomal protein S6 in vitro. The complete characterization and purification of this enzyme may be essential to the elucidation of the mechanism of regulation of S6 phosphorylation by insulin.     

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.     

Epidermal growth factor-mediated activation of an S6 kinase in Swiss mouse 3T3 cells

Extracts from epidermal growth factor (EGF)-stimulated Swiss mouse 3T3 cells are up to 10 times more potent in phosphorylating ribosomal protein S6 than extracts from quiescent cells. Preparation of extracts in the absence of phosphatase inhibitors leads to a time-dependent loss of kinase activity. In order of potency, the most efficient phosphatase inhibitors in protecting the S6 kinase activity are phosphotyrosine followed by p-nitrophenyl phosphate, beta-glycerol phosphate, and phosphoserine. The kinetics of kinase activation following EGF treatment are rapid and transient. The maximum increase is observed between 15 and 30 min with only 20-30% of the activity remaining after 2 h. Phosphorylation of S6 in the intact cell follows a similar pattern of activation, reaching a maximum between 30 and 60 min and then slowly returning to basal levels by approximately 3 h. The activation of protein synthesis is also rapid; however, in contrast to the transient activation of the S6 kinase and S6 phosphorylation, it remains persistently high for at least 6 h following EGF treatment. Comparison of these events with EGF binding shows that about 50% of the cell surface binding sites are lost within 10 min of exposure to EGF, and about 25% remain after 2 h. Finally, sodium orthovanadate, which is known to mimic the mitogenic effect of EGF, also leads to activation of the S6 kinase, however, with distinct kinetics and by an apparent EGF receptor-independent pathway.     

Identification of the NIMA family kinases NEK6/7 as regulators of the p70 ribosomal S6 kinase

BACKGROUND: The p70 S6 kinase, like several other AGC family kinases, requires for activation the concurrent phosphorylation of a site on its activation loop and a site carboxyterminal to the catalytic domain, situated in a hydrophobic motif site FXXFS/TF/Y, e.g.,Thr412 in p70 S6 kinase (alpha 1). Phosphorylation of the former site is catalyzed by PDK1, whereas the kinase responsible for the phosphorylation of the latter site is not known. RESULTS: The major protein kinase that is active on the p70 S6 kinase hydrophobic regulatory site, Thr412, was purified from rat liver and identified as the NIMA-related kinases NEK6 and NEK7. Recombinant NEK6 phosphorylates p70 S6 kinase at Thr412 and other sites and activates the p70 S6 kinase in vitro and in vivo, in a manner synergistic with PDK1. Kinase-inactive NEK6 interferes with insulin activation of p70 S6 kinase. The activity of recombinant NEK6 is dependent on its phosphorylation, but NEK6 activity is not regulated by PDK1 and is only modestly responsive to insulin and PI-3 kinase inhibitors. CONCLUSION: NEK6 and probably NEK7 are novel candidate physiologic regulators of the p70 S6 kinase.     

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.     

Interleukin 2 and diacylglycerol stimulate phosphorylation of 40 S ribosomal S6 protein. Correlation with increased protein synthesis and S6 kinase activation

Interleukin 2 (IL-2) and the synthetic diacylglycerol, 1-oleoyl-2-acetylglycerol (OAG), a direct activator of protein kinase C, induce phosphorylation of the ribosomal S6 protein in a murine IL-2-dependent lymphocyte clone. The phosphorylation of S6 protein was correlated with increased protein synthesis in this cell line. Using cell-free assay systems, two unique kinases capable of phosphorylating the S6 protein were identified, namely, a calcium/phospholipid-dependent phosphotransferase, protein kinase C, and a second phospholipid-independent kinase detected in crude cytosolic fractions. Peptide mapping of the S6 protein demonstrated that the degree of S6 phosphorylation stimulated by IL-2 and OAG was similar to that achieved using the second (calcium/phospholipid-independent) kinase but not to the level of phosphorylation achieved with protein kinase C. The kinase responsible for phosphorylating S6 was soluble in stimulated cells and was induced in a time-dependent manner by either IL-2 or diacylglycerol treatment of intact cells. These data support the notion that, although protein kinase C is activated by IL-2 or OAG, subsequent events such as S6 phosphorylation may be the result of the activation of secondary phosphotransferase systems regulated by protein kinase C.     

EGF induces biphasic S6 kinase activation: late phase is protein kinase C-dependent and contributes to mitogenicity

Detailed kinetics reveal that EGF-induced S6 kinase activation is biphasic: an early phase appears at 10-15 min, followed by a late phase between 30 and 60 min. Both activities exhibit the same chromatographic behavior and sensitivity to phosphatase 2A. Direct activation of protein kinase C by TPA induces only late phase activity. Down-regulation of protein kinase C leads to loss of both TPA- and EGF-induced late phase activity, while the early phase is unaffected. The loss of late phase kinase activity results in decreased EGF-induced S6 phosphorylation, protein synthesis, and cell growth. The results indicate that EGF differentially regulates S6 kinase activation by two distinct signaling pathways and that loss of the late or protein kinase C-dependent phase leads to a diminished mitogenic response.     

Extracellular zinc activates p70 S6 kinase through the phosphatidylinositol 3-kinase signaling pathway

We have studied a possible role of extracellular zinc ion in the activation of p70S6k, which plays an important role in the progression of cells from the G(1) to S phase of the cell cycle. Treatment of Swiss 3T3 cells with zinc sulfate led to the activation and phosphorylation of p70S6k in a dose-dependent manner. The activation of p70S6k by zinc treatment was biphasic, the early phase being at 30 min followed by the late phase at 120 min. The zinc-induced activation of p70S6k was partially inhibited by down-regulation of phorbol 12-myristate 13-acetate-responsive protein kinase C (PKC) by chronic treatment with phorbol 12-myristate 13-acetate, but this was not significant. Moreover, Go6976, a specific calcium-dependent PKC inhibitor, did not significantly inhibit the activation of p70S6k by zinc. These results demonstrate that the zinc-induced activation of p70S6k is not related to PKC. Also, extracellular calcium was not involved in the activation of p70S6k by zinc. Further characterization of the zinc-induced activation of p70S6k using specific inhibitors of the p70S6k signaling pathway, namely rapamycin, wortmannin, and LY294002, showed that zinc acted upstream of mTOR/FRAP/RAFT and phosphatidylinositol 3-kinase (PI3K), because these inhibitors caused the inhibition of zinc-induced p70S6k activity. In addition, Akt, the upstream component of p70S6k, was activated by zinc in a biphasic manner, as was p70S6k. Moreover, dominant interfering alleles of Akt and PDK1 blocked the zinc-induced activation of p70S6k, whereas the lipid kinase activity of PI3K was potently activated by zinc. Taken together, our data suggest that zinc activates p70S6k through the PI3K signaling pathway.     

An array of insulin-activated, proline-directed serine/threonine protein kinases phosphorylate the p70 S6 kinase.

This study characterizes the insulin-activated serine/threonine protein kinases in H4 hepatoma cells active on a 37-residue synthetic peptide (called the SKAIPS peptide) corresponding to a putative autoinhibitory domain in the carboxyl-terminal tail of the p70 S6 kinase as well as on recombinant p70 S6 kinase. Three peaks of insulin-stimulated protein kinase active on both these substrates are identified as two (possibly three) isoforms of the 40-45-kDa erk/microtubule-associated protein (MAP)-2 kinase family and a 150-kDa form of cdc2. Although distinguishable in their substrate specificity, these protein kinases together with the p54 MAP-2 kinase share a major common specificity determinant reflected in the SKAIPS peptide: the requirement for a proline residue immediately carboxyl-terminal to the site of Ser/Thr phosphorylation. In addition, however, at least one peak of insulin-stimulated protein kinase active on recombinant p70, but not on the SKAIPS peptide, is present although not yet identified. MFP/cdc2 phosphorylates both rat liver p70 S6 kinase and recombinant p70 S6 kinase exclusively at a set of Ser/Thr residues within the putative autoinhibitory (SKAIPS peptide) domain. erk/MAP kinase does not phosphorylate rat liver p70 S6 kinase, but readily phosphorylates recombinant p70 S6 kinase at sites both within and in addition to those encompassed by the SKAIPS peptide sequences. Although the tryptic 32P-peptides bearing the cdc2 and erk/MAP kinase phosphorylation sites co-migrate with a subset of the sites phosphorylated in situ in insulin-stimulated cells, phosphorylation of the p70 S6 kinase by these proline-directed protein kinases in vitro does not reproducibly activate p70 S6 kinase activity. Thus, one or more erk/MAP kinases and cdc2 are likely to participate in the insulin-induced phosphorylation of the p70 S6 kinase. In addition to these kinases, however, phosphorylation of the p70 S6 kinase by other as yet unidentified protein kinases is necessary to recapitulate the multisite phosphorylation required for activation of the p70 S6 kinase.     

c-Raf/MEK/ERK pathway controls protein kinase C-mediated p70S6K activation in adult cardiac muscle cells

p70S6 kinase (S6K1) plays a pivotal role in hypertrophic cardiac growth via ribosomal biogenesis. In pressure-overloaded myocardium, we show S6K1 activation accompanied by activation of protein kinase C (PKC), c-Raf, and mitogen-activated protein kinases (MAPKs). To explore the importance of the c-Raf/MAPK kinase (MEK)/MAPK pathway, we stimulated adult feline cardiomyocytes with 12-O-tetradecanoylphorbol-13-acetate (TPA), insulin, or forskolin to activate PKC, phosphatidylinositol-3-OH kinase, or protein kinase A (PKA), respectively. These treatments resulted in S6K1 activation with Thr-389 phosphorylation as well as mammalian target of rapamycin (mTOR) and S6 protein phosphorylation. Thr-421/Ser-424 phosphorylation of S6K1 was observed predominantly in TPA-treated cells. Dominant negative c-Raf expression or a MEK1/2 inhibitor (U0126) treatment showed a profound blocking effect only on the TPA-stimulated phosphorylation of S6K1 and mTOR. Whereas p38 MAPK inhibitors exhibited only partial effect, MAPK-phosphatase-3 expression significantly blocked the TPA-stimulated S6K1 and mTOR phosphorylation. Inhibition of mTOR with rapamycin blocked the Thr-389 but not the Thr-421/Ser-424 phosphorylation of S6K1. Therefore, during PKC activation, the c-Raf/MEK/extracellular signal-regulated kinase-1/2 (ERK1/2) pathway mediates both the Thr-421/Ser-424 and the Thr-389 phosphorylation in an mTOR-independent and -dependent manner, respectively. Together, our in vivo and in vitro studies indicate that the PKC/c-Raf/MEK/ERK pathway plays a major role in the S6K1 activation in hypertrophic cardiac growth.     

Ribosomal S6 kinase signaling and the control of translation

The highly homologous 40S ribosomal protein S6 kinases (S6K1 and S6K2) play a key role in the regulation of cell growth by controlling the biosynthesis of translational components which make up the protein synthetic apparatus, most notably ribosomal proteins. In the case of S6K1, at least eight phosphorylation sites are believed to mediate kinase activation in a hierarchical fashion. Activation is initiated by phosphatidylinositide-3OH kinase (PI3K)-mediated phosphorylation of key residues in the carboxy-terminus of the kinase, allowing phosphorylation of a critical residue residing in the activation loop of the catalytic domain by phosphoinositide-dependent kinase 1 (PDK1). The kinases responsible for phosphorylating the carboxy-terminal sites have yet to be identified. Additionally, S6 kinases are under the control of the PI3K relative, mammalian Target Of Rapamycin (mTOR), which may serve an additional function as a checkpoint for amino acid availability. In this review we set out to discuss the present state of knowledge regarding upstream signaling components which have been implicated in the control of S6K1 activation and the role of the kinase in controlling cell growth through regulating ribosome biogenesis at the translational level.     

Phosphorylation of ribosomal protein S6 and a peptide analogue of S6 by a protease-activated kinase isolated from rat liver

A trypsin-activated protein kinase has been isolated from rat liver using a peptide analogue of ribosomal protein S6 as a substrate in kinase assays. The structure of the peptide, Arg-Arg-Leu-Ser-Ser-Leu-Arg-Ala, was based on a region of S6 containing both an insulin- and cyclic AMP-regulated phosphorylation site. The trypsin-activated protein kinase phosphorylated a corresponding site in the peptide analogue and ribosomal protein S6 that was distinct from the preferred site for cyclic AMP-dependent protein kinase. Ribosomal S6 contained at least one other major site for the trypsin-activated protein kinase.     

Stimulation of MAP kinase and S6 kinase by vanadium and selenium in rat adipocytes

To explore the mechanism underlying the insulin-mimetic actions of vanadium and selenium we examined their effects on the mitogen activated protein/myelin basic protein kinases (MAPK) and ribosomal S6 protein kinases, which are among the best characterized of the kinases that comprise the phosphorylation cascade in insulin signal transduction. We observed a transient activation of MAPK and S6 kinases by insulin in rat adipocytes, while both sodium selenate and vanadyl sulphate produced prolonged activation of the kinases. Vanadyl sulphate stimulated the activity of MAPK and S6 kinase by as much as 6 fold and 15 fold, respectively. Pretreatment of the cells with genistein did not affect the activation of MAPK by insulin, but partially blocked the effects of sodium selenate and vanadyl sulphate. Genistein did not change the activation of S6 kinase by insulin, but blocked the activation in vanadyl sulphate- and sodium selenate-treated-cells, suggesting that a genistein sensitive tyrosine kinase may be involved in the activation by these two compounds. Rapamycin, a specific inhibitor of the p70s6k isoform of S6 kinase, partially reduced the activation of S6 kinase activity by sodium selenate, indicating a role for this kinase in the overall activity of the S6 kinase in sodium selenate-treated cells. A similar trend was noted in vanadyl sulphate-treated cells. Thus, this study supports the involvement of MAPK and S6 kinases in the insulin-mimetic actions of vanadium and selenium.