Phospholipase D1 is phosphorylated and activated by protein kinase C in caveolin-enriched microdomains within the plasma membrane

Activities of phospholipase D (PLD) in diverse subcellular organelles have been identified but the details of regulatory mechanisms in such locations are unknown. Protein kinase C (PKC) is a major regulator of PLD. Serine 2, threonine 147, and serine 561 residues of phospholipase D1 (PLD1) were determined as sites of phosphorylation by PKC (Kim, Y., Han, J. M., Park, J. B., Lee, S. D., Oh, Y. S., Chung, C., Lee, T. G., Kim, J. H., Park, S. K., Yoo, J. S., Suh, P. G., Ryu, S. H. (1999) Biochemistry 38, 10344-10351). In our present study, a triple mutation of these phosphorylation sites diminished markedly phorbol 12-myristate 13-acetate (PMA)-induced PLD1 activity in COS-7 cells. We looked at the location of the PLD1 phosphorylation by PKC by observing PMA induced band shifts and by use of anti-phospho-PLD1 monoclonal antibody. The shifted PMA-induced proteins and the immunoreactivity of the anti-phospho-PLD1 antibody were mainly found in the caveolin-enriched membrane (CEM) fraction. Depletion of cellular cholesterol led to a loss of this compartmentalization of phosphorylated PLD1 in the CEM. Replacement of the cellular cholesterol led to the restoration of phosphorylated PLD1 in the CEM. Immunocytochemical studies of COS-7 cells revealed that PLD1 was localized in the plasma membrane as well as in the vesicular structures in the cytoplasm, but the phosphorylation of PLD1 occurred only in the plasma membrane. Our results, therefore, show that phosphorylation, and thereby activation, of PLD1 by PKC occurs in the caveolin and cholesterol-enriched low density domain of the plasma membrane in COS-7 cells.     

Differential tyrosine phosphorylation of phospholipase D isozymes by hydrogen peroxide and the epidermal growth factor in A431 epidermoid carcinoma cells.

The regulatory mechanism through which the phospholipase D (PLD) isoforms PLD1 and PLD2 are activated is poorly understood. We investigated the possibility that the PLD isozymes are differentially regulated in response to pharmacologic stimulants in cells. In this report, we demonstrate for the first time that H2O2 and EGF differentially induce tyrosine phosphorylation of the PLD isozymes in A431 cells, which express both PLD1 and PLD2. H2O2 induced tyrosine phosphorylation of PLD1 and PLD2, whereas EGF only caused the tyrosine phosphorylation of PLD2. Both agents also induced phosphorylation of the EGF receptor. Interestingly, the PLD isozymes were associated with the EGF receptor and PKC-alpha in a ligand independent manner. Activation of PLD by H2O2 and EGF nearly correlated with tyrosine phosphorylation of the protein in PLD1 immune complexes. Activation of PLD by both agents was inhibited by the PKC inhibitor, Ro 31-8220, and by the down-regulation of PKC. Pretreatment of the cells with the tyrosine kinase inhibitor tyrphostin AG1478 resulted in inhibition of the H2O2 and EGF-induced tyrosine phosphorylation and PLD activation. These results indicate that H2O2 and EGF induce differential tyrosine phosphorylation of PLD isozymes. Also, the activation of PLD by these agonists involves tyrosine phosphorylation and PKC activation.     

Sites on phospholipase D2 phosphorylated by PKCalpha

The phosphorylation sites in phospholipase D2 (PLD2) induced by activation of protein kinase Calpha (PKCalpha) in COS 7 cells were analyzed by mass spectrometry. Ser134, 146, and 243, and Thr72, 99/100, and 252 were identified. These sites were mutated to Ala and the double mutation of Ser243 and Thr252 eliminated the phosphorylation. However, the PLD2 activity, and the binding between PKCalpha and PLD2 were unaffected by the mutations. We conclude that phosphorylation of these residues is not required for PLD2 activation by PKCalpha, and that protein-protein interaction between PLD2 and PKCalpha is sufficient to activate PLD2.     

Localization of phospholipase D1 to caveolin-enriched membrane via palmitoylation: implications for epidermal growth factor signaling

Phospholipase D (PLD) has been suggested to mediate epidermal growth factor (EGF) signaling. However, the molecular mechanism of EGF-induced PLD activation has not yet been elucidated. We investigated the importance of the phosphorylation and compartmentalization of PLD1 in EGF signaling. EGF treatment of COS-7 cells transiently expressing PLD1 stimulated PLD1 activity and induced PLD1 phosphorylation. The EGF-induced phosphorylation of threonine147 was completely blocked and the activity of PLD1 attenuated by point mutations (S2A/T147A/S561A) of PLD1 phosphorylation sites. The expression of a dominant negative PKCalpha mutant by adenovirus-mediated gene transfer greatly inhibited the phosphorylation and activation of PLD1 induced by EGF in PLD1-transfected COS-7 cells. EGF-induced PLD1 phosphorylation occurred primarily in the caveolin-enriched membrane (CEM) fraction, and the kinetics of PLD1 phosphorylation in the CEM were strongly correlated with PLD1 phosphorylation in the total membrane. Interestingly, EGF-induced PLD1 phosphorylation and activation and the coimmunoprecipitation of PLD1 with caveolin-1 and the EGF receptor in the CEM were significantly attenuated in the palmitoylation-deficient C240S/C241S mutant, which did not localize to the CEM. Immunocytochemical analysis revealed that wild-type PLD1 colocalized with caveolin-1 and the EGF receptor and that phosphorylated PLD1 was localized exclusively in the plasma membrane, although some PLD1 was also detected in vesicular structures. Transfection of wild-type PLD1 but not of C240S/C241S mutant increased EGF-induced raf-1 translocation to the CEM and ERK phosphorylation. This study shows, for the first time, that EGF-induced PLD1 phosphorylation and activation occur in the CEM and that the correct localization of PLD1 to the CEM via palmitoylation is critical for EGF signaling.     

Cdk5 mediates vimentin Ser56 phosphorylation during GTP-induced secretion by neutrophils

Secretion by neutrophils contributes to acute inflammation following injury or infection. Vimentin has been shown to be important for secretion by neutrophils but little is known about its dynamics during secretion, which is regulated by cyclin-dependent kinase 5 (Cdk5). In this study, we sought to examine the vimentin dynamics and its potential regulation by Cdk5 during neutrophil secretion. We show that vimentin is a Cdk5 substrate that is specifically phosphorylated at Ser56. In response to neutrophil stimulation with GTP, vimentin Ser56 was phosphorylated and colocalized with Cdk5 in the cytoplasmic compartment. Vimentin pSer56 and Cdk5 colocalization was consistent with coimmunoprecipitation from stimulated cells. Vimentin Ser56 phosphorylation occurred immediately after stimulation, and a remarkable increase in phosphorylation was noted later in the secretory process. Decreased GTP-induced vimentin Ser56 phosphorylation and secretion resulted from inhibition of Cdk5 activity by roscovitine or olomoucine or by depletion of Cdk5 by siRNA, suggesting that GTP-induced Cdk5-mediated vimentin Ser56 phosphorylation may be related to GTP-induced Cdk5-mediated secretion by neutrophils. Indeed, inhibition of vimentin Ser56 phosphorylation led to a corresponding inhibition of GTP-induced secretion, indicating a link between these two events. While fMLP also induced vimentin Ser56 phosphorylation, such phosphorylation was unaffected by roscovitine, which nonetheless, inhibited secretion, suggesting that Cdk5 regulates fMLP-induced secretion via a mechanism independent of Cdk5-mediated vimentin Ser56 phosphorylation. These findings demonstrate the distinct involvement of Cdk5 in GTP- and fMLP-induced secretion by neutrophils, and support the notion that specific targeting of Cdk5 may serve to inhibit the neutrophil secretory process.     

Activation and glucagon regulation of mitogen-activated protein kinases (MAPK) by insulin and epidermal growth factor in cultured rat and human hepatocytes

Many hepatocellular activities may be proximally regulated by intracellular signalling proteins including mitogen-activated protein kinases (MAPK). In this study, signalling events from epidermal growth factor (EGF) and insulin were examined in primary cultured human and rat hepatocytes. Using Western immunoblots, rat and human hepatocytes were found to produce a rapid tyrosine phosphorylation of the EGF receptor and MAPK following 0·5–1 min exposure to EGF. Phosphorylation of p42 and p44 MAPK was observed following 2·5 min exposure to EGF. Insulin treatment produced phosphorylation of the insulin receptor β subunit; shc phosphorylation was not observed. MAPK phosphorylation corresponded with a shift in molecular weight and an increase in kinase activity. Insulin-dependent activation of MAPK was unequivocally observed only in human hepatocytes, though a slight activation was detected in rat. Co-treatment with insulin and EGF produced phosphorylation and complete electrophoretic shift in molecular weight of MAPK, with an additive or synergistic increase in enzyme activity in rat but not human hepatocytes; human hepatocyte MAPK was maximally stimulated by EGF alone. Glucagon pretreatment blocked phosphorylation, gel mobility shift and kinase activity of MAPK induced by insulin but only partially blocked EGF-induced MAPK activation in human hepatocytes. Glucagon also reduced the activation of MAPK by EGF in rat hepatocytes. Pre-treatments with forskolin or cyclic AMP analogues diminished in the insulin-, EGF- and insulin plus EGF-dependent activation of MAPK in rat hepatocytes without effecting phosphorylation of receptors or MAPK. These results indicate that although EGF and insulin may both signal through the MAPK/ras/raf/MAPK pathway, the response for MAPK differs between these ligands and between species. Further, in both rat and human, glucagon exerts its effects through a cyclic AMP-dependent mechanism at a level in the insulin and EGF signal transduction pathways downstream of MAPK but promixal to MAPK. The partial inhibition of EGF-induced MAPK phosphorylation by glucagon in human hepatocytes provides further evidence for a raf-1-independent pathway for activation of MAPK. © 1998 John Wiley & Sons, Ltd.     

Actin directly interacts with phospholipase D, inhibiting its activity

Mammalian phospholipase D (PLD) plays a key role in several signal transduction pathways and is involved in many diverse functions. To elucidate the complex molecular regulation of PLD, we investigated PLD-binding proteins obtained from rat brain extract. Here we report that a 43-kDa protein in the rat brain, beta-actin, acts as a major PLD2 direct-binding protein as revealed by peptide mass fingerprinting in combination with matrix-assisted laser desorption ionization/time-of-flight mass spectrometry. We also determined that the region between amino acids 613 and 723 of PLD2 is required for the direct binding of beta-actin, using bacterially expressed glutathione S-transferase fusion proteins of PLD2 fragments. Intriguingly, purified beta-actin potently inhibited both phosphatidylinositol-4,5-bisphosphate- and oleate-dependent PLD2 activities in a concentration-dependent manner (IC50 = 5 nm). In a previous paper, we reported that alpha-actinin inhibited PLD2 activity in an interaction-dependent and an ADP-ribosylation factor 1 (ARF1)-reversible manner (Park, J. B., Kim, J. H., Kim, Y., Ha, S. H., Kim, J. H., Yoo, J.-S., Du, G., Frohman, M. A., Suh, P.-G., and Ryu, S. H. (2000) J. Biol. Chem. 275, 21295-21301). In vitro binding analyses showed that beta-actin could displace alpha-actinin binding to PLD2, demonstrating independent interaction between cytoskeletal proteins and PLD2. Furthermore, ARF1 could steer the PLD2 activity in a positive direction regardless of the inhibitory effect of beta-actin on PLD2. We also observed that beta-actin regulates PLD1 and PLD2 with similar binding and inhibitory potencies. Immunocytochemical and co-immunoprecipitation studies demonstrated the in vivo interaction between the two PLD isozymes and actin in cells. Taken together, these results suggest that the regulation of PLD by cytoskeletal proteins, beta-actin and alpha-actinin, and ARF1 may play an important role in cytoskeleton-related PLD functions.     

Chk1 phosphorylates the tumour suppressor Mig-6, regulating the activation of EGF signalling

The tumour suppressor gene product Mig-6 acts as an inhibitor of epidermal growth factor (EGF) signalling. However, its posttranslational modifications and regulatory mechanisms have not been elucidated. Here, we investigated the phosphorylation of human Mig-6 and found that Chk1 phosphorylated Mig-6 in vivo as well as in vitro. Moreover, EGF stimulation promoted phosphorylation of Mig-6 without DNA damage and the phosphorylation was inhibited by depletion of Chk1. EGF also increased Ser280-phosphorylated Chk1, a cytoplasmic-tethering form, via PI3K pathway. Mass spectrometric analyses suggested that Ser 251 of Mig-6 was a major phosphorylation site by Chk1 in vitro and in vivo. Substitution of Ser 251 to alanine increased inhibitory activity of Mig-6 against EGF receptor (EGFR) activation. Moreover, EGF-dependent activation of EGFR and cell growth were inhibited by Chk1 depletion, and were rescued by co-depletion of Mig-6. Our results suggest that Chk1 phosphorylates Mig-6 on Ser 251, resulting in the inhibition of Mig-6, and that Chk1 acts as a positive regulator of EGF signalling. This is a novel function of Chk1.     

Intracellular signaling of angiotensin II-induced p70 S6 kinase phosphorylation at Ser(411) in vascular smooth muscle cells. Possible requirement of epidermal growth factor receptor, Ras, extracellular signal-regulated kinase, and Akt

Activation of p70 S6 kinase (p70(S6K)) by growth factors requires multiple signal inputs involving phosphoinositide 3-kinase (PI3K), its effector Akt, and an unidentified kinase that phosphorylates Ser/Thr residues (Ser(411), Ser(418), Ser(424), and Thr(421)) clustered at its autoinhibitory domain. However, the mechanism by which G protein-coupled receptors activate p70(S6K) remains largely uncertain. By using vascular smooth muscle cells in which we have demonstrated Ras/extracellular signal-regulated kinase (ERK) activation through Ca(2+)-dependent, epidermal growth factor (EGF) receptor transactivation by G(q)-coupled angiotensin II (Ang II) receptor, we present a unique cross-talk required for Ser(411) phosphorylation of p70(S6K) by Ang II. Both p70(S6K) Ser(411) and Akt Ser(473) phosphorylation by Ang II appear to involve EGF receptor transactivation and were inhibited by dominant-negative Ras, whereas the phosphorylation of p70(S6K) and ERK but not Akt was sensitive to the MEK inhibitor. By contrast, the phosphorylation of p70(S6K) and Akt but not ERK was sensitive to PI3K inhibitors. Similar inhibitory pattern on these phosphorylation sites by EGF but not insulin was observed. Taken together with the inhibition of Ang II-induced p70(S6K) activation by dominant-negative Ras and the MEK inhibitor, we conclude that Ang II-initiated activation of p70(S6K) requires both ERK cascade and PI3K/Akt cascade that bifurcate at the point of EGF receptor-dependent Ras activation.     

Cdk5-dependent regulation of glucose-stimulated insulin secretion

Tight glycemic control in individuals with diabetes mellitus is essential to prevent or delay its complications. Present treatments to reduce hyperglycemia mainly target the ATP-sensitive K(+) (K(ATP)) channel of pancreatic beta cells to increase insulin secretion. These current approaches are often associated with the side effect of hypoglycemia. Here we show that inhibition of the activity of cyclin-dependent kinase 5 (Cdk5) enhanced insulin secretion under conditions of stimulation by high glucose but not low glucose in MIN6 cells and pancreatic islets. The role of Cdk5 in regulation of insulin secretion was confirmed in pancreatic beta cells deficient in p35, an activator of Cdk5. p35-knockout mice also showed enhanced insulin secretion in response to a glucose challenge. Cdk5 kinase inhibition enhanced the inward whole-cell Ca(2+) channel current and increased Ca(2+) influx across the L-type voltage-dependent Ca(2+) channel (L-VDCC) upon stimulation with high glucose in beta cells, but had no effect on Ca(2+) influx without glucose stimulation. The inhibitory regulation by Cdk5 on the L-VDCC was attributed to the phosphorylation of loop II-III of the alpha(1C) subunit of L-VDCC at Ser783, which prevented the binding to SNARE proteins and subsequently resulted in a decrease of the activity of L-VDCC. These results suggest that Cdk5/p35 may be a drug target for the regulation of glucose-stimulated insulin secretion.     

The death effector domain-containing DEDD forms a complex with Akt and Hsp90, and supports their stability

Insulin secretion and glucose transport are the major mechanisms to balance glucose homeostasis. Recently, we found that the death effector domain-containing DEDD inhibits cyclin-dependent kinase-1 (Cdk1) function, thereby preventing Cdk1-dependent inhibitory phosphorylation of S6 kinase-1 (S6K1), downstream of phosphatidylinositol 3-kinase (PI3K), which overall results in maintenance of S6K1 activity. Here we newly show that DEDD forms a complex with Akt and heat-shock protein 90 (Hsp90), and supports the stability of both proteins. Hence, in DEDD^−/− mice, Akt protein levels are diminished in skeletal muscles and adipose tissues, which interferes with the translocation of glucose-transporter 4 (GLUT4) upon insulin stimulation, leading to inefficient incorporation of glucose in these organs. Interestingly, as for the activation of S6K1, suppression of Cdk1 is involved in the stabilization of Akt protein by DEDD, since diminishment of Cdk1 in DEDD^−/− cells via siRNA expression or treatment with a Cdk1-inhibitor, increases both Akt and Hsp90 protein levels. Such multifaceted involvement of DEDD in glucose homeostasis by supporting both insulin secretion (via maintenance of S6K1 activity) and glucose uptake (via stabilizing Akt protein), may suggest an association of DEDD-deficiency with the pathogenesis of type 2 diabetes mellitus.     

Tyrosine residues in bovine phospholipase C-gamma phosphorylated by the epidermal growth factor receptor in vitro.

We have identified the sites phosphorylated in vitro by epidermal growth factor (EGF) receptor kinase in bovine brain phospholipase C-gamma (PLC-gamma). They are tyrosine residues 472, 771, 783, and 1254. The rate of phosphorylation was fastest with the sites at 771 and 783, then at 1254, and slowest at 472. PLC-gamma isolated from cells treated with EGF is known to contain at least four tyrosine phosphate-containing peptides and two of them are identified to be residues 771 and 1254 in the accompanying paper (Wahl, M. I., Nishibe, S., Kim, J. W., Kim, H., Rhee, S. G., and Carpenter, G. (1990) J. Biol. Chem. 265, 3944-3948). The 3 residues 472, 771, and 783 are located closely to the regions of PLC-gamma which exhibit a high sequence similarity to the regulatory domain of the src family tyrosine kinases. Nevertheless, the tyrosine phosphorylation did not affect the catalytic activity of PLC-gamma in vitro. We propose, therefore, that the phosphorylation of PLC-gamma by EGF receptor kinase alters its interaction with putative inhibitory proteins and leads to its activation.     

Interleukin-6 impairs the insulin signaling pathway, promoting production of nitric oxide in human umbilical vein endothelial cells

Interleukin 6 (IL-6) is an independent predictor of type 2 diabetes and cardiovascular disease and is correlated with insulin resistance. Insulin stimulates nitric oxide (NO) production through the IRS-1/PI3-kinase/Akt/eNOS pathway (where IRS-1 is insulin receptor substrate 1, PI3-kinase is phosphatidylinositol 3-kinase, and eNOS is endothelial NO synthase). We asked if IL-6 affects insulin vasodilator action both in human umbilical vein endothelial cells (HUVEC) and in the aortas of C57BL/6J mice and whether this inhibitory effect was caused by increased Ser phosphorylation of IRS-1. We observed that IL-6 increased IRS-1 phosphorylation at Ser(312) and Ser(616); these effects were paralleled by increased Jun N-terminal protein kinase (JNK) and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation and reversed by JNK and ERK1/2 inhibition. In addition, IL-6 treatment resulted in impaired IRS-1 phosphorylation at Tyr(612), a site essential for engaging PI3-kinase. Furthermore, IL-6 treatment reduced insulin-stimulated phosphorylation of eNOS at the stimulatory Ser(1177) site and impaired insulin-stimulated eNOS dephosphorylation at the inhibitory Thr(495) site. Insulin-stimulated eNOS activation and NO production were also inhibited by IL-6; these effects were reversed by inhibition of JNK and ERK1/2. Treatment of C57BL/6J mice with IL-6 resulted in impaired insulin-dependent activation of the Akt/eNOS pathway in the aorta as a result of JNK and ERK1/2 activation. Our data suggest that IL-6 impairs the vasodilator effects of insulin that are mediated by the IRS-1/PI3-kinase/Akt/eNOS pathway through activation of JNK and ERK1/2.     

Identification and structure characterization of a Cdk inhibitory peptide derived from neuronal-specific Cdk5 activator

The activation of cyclin-dependent kinase 5 (Cdk5) depends on the binding of its neuronal specific activator Nck5a. The minimal activation domain of Nck5a is located in the region of amino acid residues 150 to 291 (Tang, D., Chun, A. C. S., Zhang, M., and Wang, J. H. (1997) J. Biol. Chem. 272, 12318-12327). In this work we show that a 29-residue peptide, denoted as the alphaN peptide, encompassing amino acid residues Gln145 to Asp173 of Nck5a is capable of binding Cdk5 to result in kinase inhibition. This peptide also inhibits an active phospho-Cdk2-cyclin A complex, with a similar potency. Direct competition experiments have shown that this inhibitory peptide does not compete with Nck5a or cyclin A for Cdk5 or Cdk2, respectively. Steady state kinetic analysis has indicated that the alphaN peptide acts as a non-competitive inhibitor of Cdk5. Nck5a complex with respect to the peptide substrate. To understand the molecular basis of kinase inhibition by the peptide, we determined the structure of the peptide in solution by circular dichroism and two-dimensional 1H NMR spectroscopy. The peptide adopts an amphipathic alpha-helical structure from residues Ser149 to Arg162 which can be further stabilized by the helix-stabilizing solvent trifluoroethanol. The hydrophobic face of the helix is likely to be the kinase binding surface.     

c-Jun N-terminal kinase (JNK) mediates feedback inhibition of the insulin signaling cascade

Activation of the c-Jun N-terminal kinase (JNK) by proinflammatory cytokines inhibits insulin signaling, at least in part, by stimulating phosphorylation of rat/mouse insulin receptor substrate 1 (Irs1) at Ser(307) (Ser(312) in human IRS1). Here we show that JNK mediated feedback inhibition of the insulin signal in mouse embryo fibroblasts, 3T3-L1 adipocytes, and 32D(IR) cells. Insulin stimulation of JNK activity required phosphatidylinositol 3-kinase and Grb2 signaling. Moreover, activation of JNK by insulin was inhibited by a cell-permeable peptide that disrupted the interaction of JNK with cellular proteins. However, the direct binding of JNK to Irs1 was not required for its activation by insulin, whereas direct binding was required for Ser(307) phosphorylation of Irs1. Insulin-stimulated Ser(307) phosphorylation was reduced 80% in cells lacking JNK1 and JNK2 or in cells expressing a mutant Irs1 protein lacking the JNK binding site. Reduced Ser(307) phosphorylation was directly related to increased insulin-stimulated tyrosine phosphorylation, Akt phosphorylation, and glucose uptake. These results support the hypothesis that JNK is a negative feedback regulator of insulin action by phosphorylating Ser(307) in Irs1.     

Role of extracellular electrolytes in the activation of ribosomal protein S6 kinase by epidermal growth factor, insulin-like growth factor 1, and insulin in ZR-75-1 cells

Activation of ribosomal protein S6 kinase by epidermal growth factor (EGF), insulin, and insulin-like growth factor 1 (IGF1) was studied in the human mammary tumor cell line ZR-75-1 in isotonic buffers. In contrast to growth factor-dependent S6 phosphorylation which is strongly dependent on extracellular pH (Chambard, J. C., and J. Pouyssegur. 1986. Exp. Cell Res. 164:282-294.) preincubation of cells in buffers with different pH values ranging from 7.5 to 6.5 had no effect on basal or EGF-stimulated S6 kinase activity. Replacement of extracellular Na+ with choline or replacement of extracellular Ca++ with EGTA also did not inhibit stimulation of S6 kinase by EGF. When intracellular Ca++ was buffered with the permeable Ca++ chelator quin2, EGF stimulation was reduced 50%. A similar inhibition of the EGF response was observed when cells were incubated in buffers with high K+ concentrations or in the presence of the K+ ionophore valinomycin. Insulin and IGF1 stimulation of S6 kinase were also inhibited by high K+ concentrations and by buffering intracellular Ca++. In contrast to the responses to EGF, insulin- and IGF1-activation of S6 kinase was enhanced when glucose was present and depended on the presence of bicarbonate in the medium. The results indicate that ionic signals generated by growth factors and insulin, such as increases in intracellular pH or Na+, do not seem to be involved in the activation of S6 kinase. However, effects of growth factors or insulin on membrane potential and/or K+ fluxes and redistribution of intracellular Ca++ may play a role in the activation process. Furthermore, the mechanism of insulin activation of S6 kinase is distinct from the growth factors by its dependency on extracellular bicarbonate.     

Insulin potentiates EGFR activation and signaling in fibroblasts

Insulin is an essential hormone for cell growth and potentiates the mitogenic actions of multiple growth factors, including EGF. While potentiation has been shown to be mediated by the upregulation of the cyclin/CDK system, the upstream mechanisms of such synergy have not been elucidated. Our study has examined whether insulin could mediate synergy by enhancing early signaling events of the EGF receptor (EGFR). Tyrosine phosphorylation at the cell periphery of confluent Swiss 3T3 fibroblasts induced by EGF was potentiated by insulin within 2 min of stimulation. Insulin potentiation of EGF-mediated phosphorylation of the EGFR occurred 2 min after stimulation. EGFR transactivation by insulin was not observed. In addition, downstream mitogenic signaling events including ERK1/2 activation and Elk-1 phosphorylation were enhanced in response to insulin and EGF coadministration. This study shows mitogenic synergy between insulin and EGF can occur at the earliest signaling event, receptor phosphorylation, and independent of transactivation.     

Oxidative stress employs phosphatidyl inositol 3-kinase and ERK signalling pathways to activate cAMP phosphodiesterase-4D3 (PDE4D3) through multi-site phosphorylation at Ser239 and Ser579

RAW macrophages, which express the PDE4D3 and PDE4D5 cAMP phosphodiesterase isoforms, exhibited increased PDE4 activity when challenged with H2O2 in a fashion that was negated by treatment with the cell permeant antioxidant, N-acetyl cysteine and by diphenyleneiodonium chloride, an inhibitor of NADPH oxidase. In Cos1 cells transfected to express PDE4D3, challenge with H2O2 caused a rapid increase in both the activity and phosphorylation of PDE4D3. Lysates from H2O2-treated COS cells caused the phosphorylation of purified, recombinant PDE4D3 at two sites. One was the established ERK phosphorylation site at Ser579, located at the extreme C-terminus of the catalytic unit, and the other was a novel site at Ser239, located at the extreme N-terminus of the catalytic unit. Double Ser239Ala:Ser579Ala mutation of PDE4D3 prevented its H2O2-dependent phosphorylation both in vitro and in intact COS cells. Phosphorylation of PDE4D3 at Ser579 was ablated by treating COS cells with the MEK inhibitor, PD98059, which also negated activation. The activity of the Ser239Ala:Ser579Ala double mutant, and the Ser579Ala single PDE4D3 mutant was unaffected by H2O2 challenge of COS cells, whilst the Ser239Ala mutant was inhibited. Wortmannin inhibited the H2O2-dependent phosphorylation of PDE4D3 in COS cells by around 50%, whilst it fully ablated phosphorylation at Ser239 as well as ablating activation of PDE4D3. Neither immunodepletion of p70S6 kinase nor siRNA-mediated knockdown of mTor inhibited the H2O2-dependent phosphorylation of PDE4D3 at Ser239. Activation of PDE4D3 by challenge with H2O2 was not additive with activation through protein kinase A (PKA)-mediated phosphorylation of PDE4D3. Challenge with H2O2 did not alter PKA-mediated phosphorylation of PDE4D3 at Ser54. H2O2 dependent phosphorylation of PDE4D3, at Ser239 and Ser579, did not alter the sensitivity of PDE4D3 to inhibition by the selective PDE4 inhibitor, rolipram. An unknown protein kinase acting downstream of phosphatidyl inositol 3-kinase phosphorylates PDE4D3 at Ser239. This switches the effect of phosphorylation by ERK at Ser579 from inhibition to activation. We propose that phosphorylation at Ser239 attenuates interaction between either UCR2 or the UCR1/UCR2 module and the PDE4 catalytic unit so as to re-programme the functional outcome effect of phosphorylation by ERK. We identify a novel process through which reactive oxygen species activate long PDE4 isoforms so as to reduce cAMP levels and thereby promote inflammatory responses.     

Dependence of EGF receptor and STAT factor activation on redox of A431 cells

Reactive oxygen species (ROS) were established to play an important role in cellular signaling as second messengers by integrating different pathways. Recently, we showed that EGF initiated a rapid tyrosine phosphorylation of both EGF-receptor and STAT factors with simultaneous increase in the intracellular ROS level. Now, we have investigated the effect of intracellular red-ox state on EGF- and H2O2-induced activation of EGF receptor, STAT1 and STAT3. We demonstrated that the pretreatment of A431 cells with antioxidant N-acetyl-L-cysteine (NAC) partly reduced the level of EGF-induced phosphorylation of proteins under investigation. Besides, H2O2-induced activation of EGF receptor, and STAT factors was fully prevented by NAC pretreatment. The inhibition of ROS generation by DPI declined EGF-dependent activation of EGF receptor and STAT factors to basal level. Our results demonstrate the essential role of cellular red-ox status in the modulation of EGF-mediated activation of receptor and STAT factors. We have postulated that EGF-induced ROS generation is a very important initial event promoting physiological activation of EGF receptor and subsequent STAT factor activation.