Ionophore A23187-induced protein-tyrosine phosphorylation of human platelets: possible synergism between Ca2+ mobilization and protein kinase C activation.

Addition of ionophore A23187 to washed human platelets caused a time- and dose-dependent increase in the phosphotyrosyl content of 135, 124 and 76 kDa proteins. Platelets loaded with intracellular Ca2+ chelator 5,5'-dimethyl-bis-(o-aminophenoxy)-ethane-N, N, N', N'-tetraacetic acid before addition of A23187 exhibited no protein-tyrosine phosphorylation. Replenishment of such platelets with extracellular CaCl2 restored A23187-induced protein-tyrosine phosphorylation. Upon stimulation with A23187, both aspirin and ADP scavengers-treated platelets exhibited protein-tyrosine phosphorylation without phosphoinositide hydrolysis and protein kinase C activation. These data show (a) that A23187 stimulates protein-tyrosine phosphorylation by the elevation of intracellular Ca2+, and (b) that A23187-induced protein-tyrosine phosphorylation is independent of formation of endoperoxides/thromboxane A2, released ADP, phosphoinositide hydrolysis and protein kinase C activation. Furthermore, a synergistic effect of A23187 and protein kinase C activators in stimulating protein-tyrosine phosphorylation is suggested.     

Inhibition of Src family kinases blocks epidermal growth factor (EGF)-induced activation of Akt, phosphorylation of c-Cbl, and ubiquitination of the EGF receptor

Stimulation of T47D cells with epidermal growth factor (EGF) results in the activation of the intrinsic tyrosine kinases of the receptor and the phosphorylation of multiple cellular proteins including the receptor, scaffold molecules such as c-Cbl, adapter molecules such as Shc, and the serine/threonine protein kinase Akt. We demonstrate that EGF stimulation of T47D cells results in the activation of the Src protein-tyrosine kinase and that the Src kinase inhibitor PP1 blocks the EGF-induced phosphorylation of c-Cbl but not the activation/phosphorylation of the EGF receptor itself. PP1 also blocks EGF-induced ubiquitination of the EGF receptor, which is presumably mediated by phosphorylated c-Cbl. Src is associated with c-Cbl, and we have previously demonstrated that the Src-like kinase Fyn can phosphorylate c-Cbl at a preferred binding site for the p85 subunit of phosphatidylinositol 3'-kinase. PP1 treatment blocks EGF-induced activation of the anti-apoptotic protein kinase Akt suggesting that Src may regulate activation of Akt, perhaps by a Src --> c-Cbl --> phosphatidylinositol 3'-kinase --> Akt pathway.     

Effects of protein kinase C activation after epidermal growth factor binding on epidermal growth factor receptor phosphorylation

The possible role of epidermal growth factor (EGF) receptor phosphorylation at threonine 654 in modulating the protein-tyrosine kinase activity of EGF-treated A431 cells has been studied. It has been suggested that EGF could indirectly activate a protein-serine/threonine kinase, protein kinase C, that can phosphorylate the EGF receptor at threonine 654. Protein kinase C is known to be activated, and threonine 654 is phosphorylated, when A431 cells are exposed to 12-O-tetradecanoylphorbol-13-acetate (TPA). The protein-tyrosine kinase activity of EGF receptors is normally evidenced in EGF-treated cells by phosphorylation of the receptor at tyrosine. This is inhibited when TPA-treated cells are exposed to EGF. We now show that receptor phosphorylation at threonine 654 can also be detected in EGF-treated A431 cells, presumably due to indirect stimulation of protein kinase C or a similar kinase. Some receptor molecules are phosphorylated both at threonine 654 and at tyrosine. Since prior phosphorylation at threonine 654 inhibits autophosphorylation, we propose that protein kinase C can phosphorylate the threonine 654 of autophosphorylated receptors. This provides evidence for models in which protein kinase C activation, consequent upon EGF binding, could reduce the protein-tyrosine kinase activity of the EGF receptor. Indeed, we find that 12-O-tetradecanoylphorbol-13-acetate, added 10 min after EGF, further increases threonine 654 phosphorylation and induces the loss of tyrosine phosphate from A431 cell EGF receptors.     

Evidence that 3-phosphoinositide-dependent protein kinase-1 mediates phosphorylation of p70 S6 kinase in vivo at Thr-412 as well as Thr-252

Protein kinase B and p70 S6 kinase are members of the cyclic AMP-dependent/cyclic GMP-dependent/protein kinase C subfamily of protein kinases and are activated by a phosphatidylinositol 3-kinase-dependent pathway when cells are stimulated with insulin or growth factors. Both of these kinases are activated in cells by phosphorylation of a conserved residue in the kinase domain (Thr-308 of protein kinase B (PKB) and Thr-252 of p70 S6 kinase) and another conserved residue located C-terminal to the kinase domain (Ser-473 of PKB and Thr-412 of p70 S6 kinase). Thr-308 of PKBalpha and Thr-252 of p70 S6 kinase are phosphorylated by 3-phosphoinositide-dependent protein kinase-1 (PDK1) in vitro. Recent work has shown that PDK1 interacts with a region of protein kinase C-related kinase-2, termed the PDK1 interacting fragment (PIF). Interaction with PIF converts PDK1 from a form that phosphorylates PKB at Thr-308 alone to a species capable of phosphorylating Ser-473 as well as Thr-308. This suggests that PDK1 may be the enzyme that phosphorylates both residues in vivo. Here we demonstrate that PDK1 is capable of phosphorylating p70 S6 kinase at Thr-412 in vitro. We study the effect of PIF on the ability of PDK1 to phosphorylate p70 S6 kinase. Surprisingly, we find that PDK1 bound to PIF is no longer able to interact with or phosphorylate p70 S6 kinase in vitro at either Thr-252 or Thr-412. The expression of PIF in cells prevents insulin-like growth factor 1 from inducing the activation of the p70 S6 kinase and its phosphorylation at Thr-412. Overexpression of PDK1 in cells induces the phosphorylation of p70 S6 kinase at Thr-412 in unstimulated cells, and a catalytically inactive mutant of PDK1 prevents the phosphorylation of p70 S6K at Thr-412 in insulin-like growth factor 1-stimulated cells. These observations indicate that PDK1 regulates the activation of p70 S6 kinase and provides evidence that PDK1 mediates the phosphorylation of p70 S6 kinase at Thr-412.     

Noradrenaline enhances the expression of the neuronal monocarboxylate transporter MCT2 by translational activation via stimulation of PI3K/Akt and the mTOR/S6K pathway

Monocarboxylate transporter 2 (MCT2) expression is up-regulated by noradrenaline (NA) in cultured cortical neurons via a putative but undetermined translational mechanism. Western blot analysis showed that p44/p42 mitogen-activated protein kinase (MAPK) was rapidly and strongly phosphorylated by NA treatment. NA also rapidly induced serine/threonine protein kinase from AKT virus (Akt) phosphorylation but to a lesser extent than p44/p42 MAPK. However, Akt activation persisted over a longer period. Similarly, NA induced a rapid and persistent phosphorylation of mammalian target of rapamycin (mTOR), a kinase implicated in the regulation of translation in the central nervous system. Consistent with activation of the mTOR/S6 kinase pathway, phosphorylation of the ribosomal S6 protein, a component of the translation machinery, could be observed upon treatment with NA. In parallel, it was found that the NA-induced increase in MCT2 protein was almost completely blocked by LY294002 (phosphoinositide 3-kinase inhibitor) as well as by rapamycin (mTOR inhibitor), while mitogen-activated protein kinase kinase and p38 MAPK inhibitors had much smaller effects. Taken together, these data reveal that NA induces an increase in neuronal MCT2 protein expression by a mechanism involving stimulation of phosphoinositide 3-kinase/Akt and translational activation via the mTOR/S6 kinase pathway. Moreover, considering the role of NA in synaptic plasticity, alterations in MCT2 expression as described in this study might represent an adaptation to face energy demands associated with enhanced synaptic transmission.     

Insulin-induced S6 kinase activation in HeLa cells and its reversal by hyperthermic stress

Insulin treatment of HeLa S3 cells activates an S6-phosphorylating protein kinase. Although this enzyme has chromatographic properties resembling those of described proteolytic fragments of other protein kinases, namely protein kinase C, protease-activated kinase II and histone-4 protein kinase, and although insulin has been proposed by others to cause S6 phosphorylation via proteolytic protein kinase activation, the insulin-induced increase in S6-kinase activity described here is probably not due to proteolysis. Rather, the activity indicates the existence, in HeLa cells, of an interconvertible S6 kinase, since the insulin-induced activity increase was rapidly reversed under hyperthermic stress, and since this effect of hyperthermia was itself reversible. The S6-kinase activities from serum- and from insulin-stimulated HeLa cells resemble each other closely and are likely to represent the same enzyme. The enzyme may therefore mediate both signals delivered by mitogens and the insulin signal. Analysed at an in vitro transfer of 1 mol phosphate/mol S6, this S6 kinase activity does not phosphorylate the (principal) S6 site recognized by the cAMP-dependent protein kinase.     

Regulation of the phosphoinositide cycle by Na+/H+ exchange and intracellular pH in human platelets.

We have found that thrombin-induced activation of protein kinase C (PKC) in platelets, measured by phosphorylation of the 47 kDa protein, is synergistically enhanced by the amiloride analogue ethylisopropylamiloride (EIA), a specific inhibitor of Na+/H+ exchange. This EIA effect was further synergistically enhanced by lowering intracellular pH (pHi) with either nigericin or sodium propionate, and reversed by raising pHi with monensin or ammonium chloride. The synergistic enhancement of thrombin-activated PKC by EIA plus nigericin was not observed when PKC was directly activated by phorbol esters. EIA and EIA plus nigericin caused a 3- to 6-fold increase in thrombin-induced diacylglycerol (DAG), but not phosphatidic acid (PA), production. EIA and nigericin also caused a marked increase in thrombin-induced breakdown and inhibition of resynthesis of phosphatidylinositol 4,5-bisphosphate (PIP2). In summary, we have presented evidence that inhibition of Na+/H+ exchange causes primarily a H(+)-mediated interruption of the phosphoinositide cycle in activated platelets, including the accumulation of DAG associated with the enhancement of PKC activation, the inhibition of conversion of DAG to PA, and increased PIP2 breakdown. These data suggest a model in which Na+/H+ and pHi play an important regulatory role in permitting the phosphoinositide cycle to proceed in thrombin-activated platelets.     

Phosphatidylinositol 3-kinase

Currently, a central question in biology is how signals from the cell surface modulate intracellular processes. In recent years phosphoinositides have been shown to play a key role in signal transduction. Two phosphoinositide pathways have been characterized, to date. In the canonical phosphoinositide turnover pathway, activation of phosphatidylinositol-specific phospholipase C results in the hydrolysis of phosphatidylinositol 4,5-bisphospate and the generation of two second messengers, inositol 1,4,5-trisphosphate and diacylglycerol. The 3-phosphoinositide pathway involves protein-tyrosine kinase-mediated recruitment and activation of phosphatidylinositol 3-kinase, resulting in the production of phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate. The 3-phosphoinositides are not substrates of any known phospholipase C, are not components of the canonical phosphoinositide turnover pathway, and may themselves act as intracellular mediators. The 3-phosphoinositide pathway has been implicated in growth factor-dependent mitogenesis, membrane ruffling and glucose uptake. Furthermore the homology of the yeast vps34 with the mammalian phosphatidylinositol 3-kinase has suggested a role for this pathway in vesicular trafficking. In this review the different mechanisms employed by protein-tyrosine kinases to activate phosphatidylinositol 3-kinase, and its involvement in the signaling cascade initiated by tyrosine phosphorylation, are examined.     

Dissociation of protein kinase C activation and sn-1,2-diacylglycerol formation. Comparison of phosphatidylinositol- and phosphatidylcholine-derived diglycerides in alpha-thrombin-stimulated fibroblasts

Diacylglycerols (DAGs) derived from phosphatidylcholine (PC) hydrolysis have been shown to activate protein kinase C (PKC) in vitro, but it is not known whether this event occurs in response to DAGs generated via agonist-induced PC hydrolysis in intact cells. In this report we have addressed this question directly, using alpha-thrombin stimulation of IIC9 fibroblasts. PKC activation in intact cells was assessed in two ways, by measuring: 1) PKC membrane association as determined by kinase activity and Western blot analysis and 2) the phosphorylation of an endogenous PKC substrate, an 80-kDa protein. Treatment with 500 ng/ml alpha-thrombin has been shown to stimulate both phosphoinositide and PC hydrolysis, whereas treatment with 100 pg/ml alpha-thrombin stimulates only PC breakdown. Using these two conditions, we show that DAG produced from phosphoinositide, but not PC hydrolysis, is associated with the activation of PKC.     

Collagen-induced binding to human platelets of platelet-derived growth factor leading to inhibition of P43 and P20 phosphorylation

Platelet-derived growth factor (PDGF) is known to inhibit collagen-induced platelet aggregation. Collagen-induced binding of 125I-PDGF to human washed platelets was therefore investigated. It was found 1) to be time-dependent, reaching a plateau at 20 degrees C after 30 min, 2) collagen concentration-dependent, 3) specifically inhibited by unlabeled PDGF, and 4) saturable. Scatchard plot analysis showed a single class of sites with 3000 +/- 450 molecules bound/cell and an apparent KD of 1.2 +/- 0.2 10(-8) M. The effects of PDGF on collagen-induced phosphoinositide breakdown and protein phosphorylation were also investigated. At 50 ng/ml PDGF, a concentration which completely inhibited collagen-induced aggregation, the breakdown of [32P]phosphatidylinositol 4,5-biphosphate (PIP2) and [32P]phosphatidylinositol 4-phosphate (PIP) was observed, but the subsequent replenishment of [32P]PIP2 was inhibited. The same PDGF concentration totally inhibited collagen-induced phosphatidic acid formation. PDGF also completely prevented phosphorylation of P43 and P20, as a result of protein kinase C activation consecutive to phosphoinositide metabolism. These results suggest that (i) a specific PDGF receptor can be induced by collagen, and (ii) PDGF can effect the early events of collagen-induced platelet activation by inhibiting PIP2 resynthesis and P43 and P20 phosphorylation. It is concluded that PDGF might be involved in a negative feed-back control of platelet activation.     

Modulation of oxidative stress and tau phosphorylation by the mTOR activator phosphatidic acid in SH-SY5Y cells

The mammalian target of rapamycin complex 1 (mTORC1) pathway including p70(S6K) (the 70-kDa p70 S6 kinase) and S6, controls protein synthesis, has anti-apoptotic functions and can phosphorylate tau protein. mTORC1 is triggered by nutrients such as phosphatidic acid (PA). Previous experimental studies have shown that oxidative stress may down-regulate this pathway leading to neuronal death. Our results showed that in human neuroblastoma cells, PA exposure can reduce H(2)O(2)-induced apoptosis and can increase tau protein phosphorylation on Ser214 via p70(S6K) activation. These findings reveal that PA, via the mTOR kinase, can trigger tau phosphorylation on a site known to reduce paired helical filament (PHF) formation.     

Effect of genistein, a tyrosine kinase inhibitor, on U46619-induced phosphoinositide phosphorylation in human platelets

Recent evidence suggests that the agonist-induced formation of phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) via PI and PIP kinases may play an important role in transmembrane signalling. In the present work, the effect of genistein, a specific inhibitor of protein-tyrosine kinase, on phosphoinositide phosphorylation was studied in human platelets stimulated with the endoperoxide analogue, U46619. At 100 microM concentration, genistein, but not the related compounds, flavone and biochanin A, which possess only weak anti-protein-tyrosine kinase activity, significantly inhibited the U46619-induced accumulation of [3H]PIP (by 71%) and [3H]PIP2. These data suggest that phosphoinositide phosphorylation may be regulated, in part, by tyrosine phosphorylation in U46619-stimulated platelets.     

The phosphorylation of protein kinase C as a potential measure of activation.

As a means of determining the role of protein kinase C in the signal transduction from novel growth factors and hormones, we investigated the effects of well-characterized agents on the phosphorylation state of protein kinase C itself. These studies show that agents that stimulate protein kinase C either directly (phorbol esters) or indirectly through phosphatidylinositol breakdown (platelet-derived growth factor) induce an increase in the phosphorylation state of the kinase. By contrast, epidermal growth factor, which does not stimulate protein kinase C in fibroblasts, does not increase the phosphorylation state of protein kinase C, but leads to a decrease. The data suggest that the phosphorylation state of protein kinase C is dynamically controlled and can be used to provide evidence of protein kinase C activation.     

Activation of phosphatidylinositol 3-kinase in cells expressing abl oncogene variants.

A phosphoinositide kinase specific for the D-3 position of the inositol ring, phosphatidylinositol (PI) 3-kinase, associates with activated receptors for platelet-derived growth factor, insulin, and colony-stimulating factor 1, with products of the oncogenes src, fms, yes, crk, and with polyomavirus middle T antigen. Efficient fibroblast transformation by proteins of the abl and src oncogene families requires activation of their protein-tyrosine kinase activity and membrane association via an amino-terminal myristoylation. We have demonstrated that the PI 3-kinase directly associates with autophosphorylated, activated protein-tyrosine kinase variants of the abl protein. In vivo, this association leads to accumulation of the highly phosphorylated products of PI 3-kinase, PI-3,4-bisphosphate and PI-3,4,5-trisphosphate, only in myristoylated, transforming abl protein variants. Myristoylation thus appears to be required to recruit PI 3-kinase activity to the plasma membrane for in vivo activation and correlates with the mitogenicity of the abl protein variants.     

The tumor suppressor PTEN negatively regulates insulin signaling in 3T3-L1 adipocytes

PTEN is a tumor suppressor with sequence homology to protein-tyrosine phosphatases and the cytoskeleton protein tensin. PTEN is capable of dephosphorylating phosphatidylinositol 3,4, 5-trisphosphate in vitro and down-regulating its levels in insulin-stimulated 293 cells. To study the role of PTEN in insulin signaling, we overexpressed PTEN in 3T3-L1 adipocytes approximately 30-fold above uninfected or control virus (green fluorescent protein)-infected cells, using an adenovirus gene transfer system. PTEN overexpression inhibited insulin-induced 2-deoxy-glucose uptake by 36%, GLUT4 translocation by 35%, and membrane ruffling by 50%, all of which are phosphatidylinositol 3-kinase-dependent processes, compared with uninfected cells or cells infected with control virus. Microinjection of an anti-PTEN antibody increased basal and insulin stimulated GLUT4 translocation, suggesting that inhibition of endogenous PTEN function led to an increase in intracellular phosphatidylinositol 3,4,5-trisphosphate levels, which stimulates GLUT4 translocation. Further, insulin-induced phosphorylation of downstream targets Akt and p70S6 kinase were also inhibited significantly by overexpression of PTEN, whereas tyrosine phosphorylation of the insulin receptor and IRS-1 or the phosphorylation of mitogen-activated protein kinase were not affected, suggesting that the Ras/mitogen-activated protein kinase pathway remains fully functional. Thus, we conclude that PTEN may regulate phosphatidylinositol 3-kinase-dependent insulin signaling pathways in 3T3-L1 adipocytes.     

Phosphorylation of the ribosomal protein S6 during agonist-induced exocytosis in exocrine glands is catalyzed by calcium-phospholipid-dependent protein kinase (protein kinase C). Experiments with guinea pig parotid glands

The ribosomal protein S6 in exocrine cells is phosphorylated during stimulation of exocytosis by cAMP-dependent or calcium-dependent agonists. Under both conditions the same tryptic S6 phosphopeptides (termed A, B, and C) were found [Padel, Kruppa, Jahn & S?ling (1983) FEBS Lett. 159, 112-118]. Studies have now been made of the phosphorylation pattern of protein S6 from purified guinea pig parotid ribosomes following in vitro phosphorylation with calmodulin-dependent, phospholipid-dependent, and cAMP-dependent protein kinases. Only the phospholipid-dependent enzyme led to the phosphorylation of peptides A, B, and C, while the cAMP-dependent enzyme phosphorylated only peptides A and C, and the calmodulin-dependent enzyme did not phosphorylate any of the phosphopeptides found in S6 from unstimulated or stimulated intact cells. Guinea pig parotid microsomes contain substantial phospholipid-dependent protein kinase activity. Stimulation of intact parotid glands with tetradecanoylphorbol acetate led to a significant phosphorylation of S6 and a similar tryptic S6 phosphopeptide pattern as seen with carbamoylcholine. It is concluded that activation of phospholipid-dependent protein kinase is responsible for the phosphorylation of protein S6 during stimulation with calcium-dependent and cAMP-dependent secretagogues.     

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.     

Hydrophobic motif phosphorylation is not required for activation loop phosphorylation of p70 ribosomal protein S6 kinase 1 (S6K1)

p70 ribosomal protein S6 kinase 1 (S6K1) is regulated by multiple phosphorylation events. Three of these sites are highly conserved among AGC kinases (cAMP dependent Protein Kinase, cGMP dependent Protein Kinase, and Protein Kinase C subfamily): the activation loop in the kinase domain, and two C-terminal sites, the turn motif and the hydrophobic motif. The common dogma has been that phosphorylation of the hydrophobic motif primes S6K1 for the phosphorylation at the activation loop by phosphoinositide-dependent protein kinase 1 (PDK1). Here, we show that the turn motif is, in fact, phosphorylated first, the activation loop second, and the hydrophobic motif is third. Specifically, biochemical analyses of a construct of S6K1 lacking the C-terminal autoinhibitory domain as well as full-length S6K1, reveals that S6K1 is constitutively phosphorylated at the turn motif when expressed in insect cells and becomes phosphorylated in vitro by purified PDK1 at the activation loop. Only the species phosphorylated at the activation loop by PDK1 gets phosphorylated at the hydrophobic motif by mammalian target of rapamycin (mTOR) in vitro. These data are consistent with a previous model in which constitutive phosphorylation of the turn motif provides the key priming step in the phosphorylation of S6K1. The data provide evidence for regulation of S6K1, where hydrophobic motif phosphorylation is not required for PDK1 to phosphorylate S6K1 at the activation loop, but instead activation loop phosphorylation of S6K1 is required for mTOR to phosphorylate the hydrophobic motif of S6K1.     

Diverse antiapoptotic signaling pathways activated by vasoactive intestinal polypeptide, epidermal growth factor, and phosphatidylinositol 3-kinase in prostate cancer cells converge on BAD

It has been demonstrated that vasoactive intestinal polypeptide, epidermal growth factor, and chronic activation of phosphatidylinositol 3-kinase can protect prostate cancer cells from apoptosis; however, the signaling pathways that they use and molecules that they target are unknown. We report that vasoactive intestinal polypeptide, epidermal growth factor, and phosphatidylinositol 3-kinase activate independent signaling pathways that phosphorylate the proapoptotic protein BAD. Vasoactive intestinal polypeptide operated via protein kinase A, epidermal growth factor required Ras activity, and effects of phosphatidylinositol 3-kinase were predominantly mediated by Akt. BAD phosphorylation was critical for the antiapoptotic effects of each signaling pathway. None of these survival signals was able to rescue cells that express BAD with mutations in phosphorylation sites, whereas knockdown of BAD expression with small hairpin RNA rendered cells insensitive to apoptosis. Taken together, these results identify BAD as a convergence point of several antiapoptotic signaling pathways in prostate cells.