Role of Src kinase in diperoxovanadate-mediated activation of phospholipase D in endothelial cells.

We have shown earlier that oxidant-induced activation of phospholipase D (PLD) in vascular endothelial cells (ECs) is regulated by protein tyrosine kinases. To further understand the regulation of oxidant-induced PLD activation, we investigated the role of Src kinase. Treatment of bovine pulmonary artery ECs (BPAECs) with a model oxidant, diperoxovanadate (DPV), at 5 microM concentration, for 30 min, stimulated PLD activity (four- to eightfold), which was attenuated by tyrosine kinase inhibitors and by Src kinase-specific inhibitors PP-1 and PP-2, in a dose- and time-dependent fashion. Furthermore, BPAECs exposed to DPV (5 microM) for 2 min showed activation of Src kinase as observed by increased tyrosine phosphorylation and autophosphorylation in Src immunoprecipitates, which was attenuated by PP-2. Src immunoprecipitates of cell lysates from control BPAECs exhibited PLD activity in cell-free preparations, which was Arf- and Rho-sensitive and was enhanced at 2 min of DPV (5 microM) treatment. Also, Western blots of Src immunoprecipitates of control cells revealed the presence of PLD(1) and PLD(2), suggesting the association of PLD with Src kinase under basal conditions. However, exposure of cells to DPV (5 microM) for 2 min enhanced the association of PLD(2) but not PLD(1) with Src. Western blotting of immunoprecipitates of PLD(1) and PLD(2) isoforms of control BPAECs revealed the presence of Src under basal conditions and exposure of cells to DPV (5 microM) for 2 min enhanced the association of PLD(2) with Src in PLD(2) immunoprecipitates. Transient expression of a dominant negative mutant of Src in BPAECs attenuated DPV- but not TPA-induced PLD activation. In cell-free preparations, Src did not phosphorylate either PLD(1) or PLD(2) compared to protein kinase Calpha or p38 mitogen-activated protein kinase. These data show for the first time a direct association of Src with PLD in ECs and regulation of PLD in intact cells.     

Role of Ca2+ in diperoxovanadate-induced cytoskeletal remodeling and endothelial cell barrier function

Diperoxovanadate (DPV), a potent inhibitor of protein tyrosine phosphatases and activator of tyrosine kinases, alters endothelial barrier function via signaling pathways that are incompletely understood. One potential pathway is Src kinase-mediated tyrosine phosphorylation of proteins such as cortactin that regulate endothelial cell (EC) cytoskeleton assembly. As DPV modulates endothelial cell signaling via protein tyrosine phosphorylation, we determined the role of DPV-induced intracellular free calcium concentration ([Ca2+]i) in activation of Src kinase, cytoskeletal remodeling, and barrier function in bovine pulmonary artery endothelial cells (BPAECs). DPV in a dose- and time-dependent fashion increased [Ca2+]i, which was partially blocked by the calcium channel blockers nifedipine and Gd3+. Treatment of cells with thapsigargin released Ca2+ from the endoplasmic reticulum, and subsequent addition of DPV caused no further change in [Ca2+]i. These data suggest that DPV-induced [Ca2+]i includes Ca release from the endoplasmic reticulum and Ca influx through store-operated calcium entry. Furthermore, DPV induced an increase in protein tyrosine phosphorylation, phosphorylation of Src and cortactin, actin remodeling, and altered transendothelial electrical resistance in BPAECs. These DPV-mediated effects were significantly attenuated by BAPTA (25 microM), a chelator of [Ca2+]i. Immunofluorescence studies reveal that the DPV-mediated colocalization of cortactin with peripheral actin was also prevented by BAPTA. Chelation of extracellular Ca2+ by EGTA had marginal effects on DPV-induced phosphorylation of Src and cortactin; actin stress fibers formation, however, affected EC barrier function. These data suggest that DPV-induced changes in [Ca2+]i regulate endothelial barrier function using signaling pathways that involve Src and cytoskeleton remodeling.     

Involvement of c-Src in diperoxovanadate-induced endothelial cell barrier dysfunction

Reactive oxygen species (ROS) generated by activated leukocytes play an important role in the disruption of endothelial cell (EC) integrity, leading to barrier dysfunction and pulmonary edema. Although ROS modulate cell signaling, information remains limited regarding the mechanism(s) of ROS-induced EC barrier dysfunction. We utilized diperoxovanadate (DPV) as a model agent to explore the role of tyrosine phosphorylation in the regulation of EC barrier function. DPV disrupted EC barrier function in a dose-dependent manner. Tyrosine kinase inhibitors, genistein, and PP-2, a specific inhibitor of Src, reduced the DPV-mediated barrier dysfunction. Consistent with these results, DPV-induced Src activation was attenuated by PP-2. Furthermore, DPV increased the association of Src with cortactin and myosin light chain kinase, indicating their potential role as cytoskeletal targets for Src. Transient overexpression of either wild-type Src or a constitutively active Src mutant potentiated the DPV-mediated decline in barrier dysfunction, whereas a dominant negative Src mutant attenuated the response. These studies provide the first direct evidence for Src involvement in DPV-induced EC barrier dysfunction.     

Regulation of phospholipase D by tyrosine kinases

Activation of phospholipase D (PLD) represents part of an important signalling pathway in mammalian cells, Phospholipase D catalyzed hydrolysis of phospholipids generates phosphatidic acid (PA) which is subsequently metabolized to lyso-PA (LPA) or diacylglycerol (DAG). While DAG is an endogenous activator of protein kinase C (PKC), PA and LPA have been recognized as second messengers as well, Activation of PLD in response to an external stimulus may involve PKC, Ca²⁺, G-proteins and/or tyrosine kinases. In this review, we will address the role of protein tyrosine phosphorylation in growth factor-, agonist- and oxidant-mediated activation of PLD. Furthermore, a possible link between PKC, Ca²⁺, G-proteins and tyrosine kinases is discussed to indicate the complexity involved in the regulation of PLD in mammalian cells.     

Angiotensin II stimulates ERK via two pathways in epithelial cells: protein kinase C suppresses a G-protein coupled receptor-EGF receptor transactivation pathway.

In GN4 rat liver epithelial cells, angiotensin II (Ang II) produces intracellular calcium and protein kinase C (PKC) signals and stimulates ERK and JNK activity. JNK activation appears to be mediated by a calcium-dependent tyrosine kinase (CADTK). To define the ERK pathway, we established GN4 cells expressing an inhibitory Ras(N17). Induction of Ras(N17) blocked EGF- but not Ang II- or phorbol ester (TPA)-dependent ERK activation. In control cells, Ang II and TPA produced minimal increases in Ras-GTP level and Raf kinase activity. PKC depletion by chronic TPA exposure abolished TPA-dependent ERK activation but failed to diminish the effect of Ang II. In PKC-depleted cells, Ang II increased Ras-GTP level and activated Raf and ERK in a Ras-dependent manner. In PKC depleted cells, Ang II stimulated Shc and Cbl tyrosine phosphorylation, suggesting that without PKC, Ang II activates another tyrosine kinase. PKC-depletion did not alter Ang II-dependent tyrosine phosphorylation or activity of p125(FAK), CADTK, Fyn or Src, but PKC depletion or incubation with GF109203X resulted in Ang II-dependent EGF receptor tyrosine phosphorylation. In PKC-depleted cells, EGF receptor-specific tyrosine kinase inhibitors blocked Ang II-dependent EGF receptor and Cbl tyrosine phosphorylation, and ERK activation. In summary, Ang II can activate ERK via two pathways; the latent EGF receptor, Ras-dependent pathway is equipotent to the Ras-independent pathway, but is masked by PKC action. The prominence of this G-protein coupled receptor to EGF receptor pathway may vary between cell types depending upon modifiers such as PKC.     

Regulation of phospholipase D

Phospholipase D (PLD) is a widely distributed enzyme that is under elaborate control by hormones, neurotransmitters, growth factors and cytokines in mammalian cells. Protein kinase C (PKC) plays a major role in the regulation of the PLD1 isozyme through interaction with its N-terminus. PKC activates this isozyme by a non-phosphorylation mechanism in vitro, but phosphorylation plays a role in the action of PKC on the enzyme in vivo. Although PLD1 can be phosphorylated by PKC in vitro, it is unclear that this occurs in vivo. Small GTPases of the ADP-ribosylation factor (ARF) and Rho families directly activate PLD1 in vitro and there is evidence that Rho proteins are involved in agonist regulation of PLD1 in vivo. ARF proteins stimulate PLD activity in the Golgi apparatus, but the role of these proteins in agonist regulation of the enzyme is less clear. PLD1 undergoes tyrosine phosphorylation in response to H₂O₂ treatment of cells. The functional consequence of this phosphorylation and soluble tyrosine kinase(s) involved are presently unknown.     

The Naturally Occurring PKC Inhibitor Sphingosine and Tumor Promoter Phorbol Ester Potentially Induce Tyrosine Phosphorylation/Activation of Oncogenic Proline-Directed Protein Kinase FA/GSK-3α in a Common Signalling Pathway

When serum-starved A431 cells were treated with 200 nM phorbol ester TPA for 15 min, the cellular activity of protein kinase F_A/glycogen synthase kinase-3 (kinase F_A/GSK-3) could be decreased to ~25% of control. Conversely, when treated with 1 M TPA for 24 hr, the activity could be reversibly increased to ~200% of Control. The naturally occurring protein kinase C (PKC) inhibitor sphingosine at a concentration of 27 M could also induce activation of kinase F_A/GSK-3 to ~200% of control within 60 min. Further, when cells were chronically treated with 1 M TPA for 24 hr and then with 27 M sphingosine for 60 min, the activity of kinase F_A/GSK-3 could only be increased to ~200% of control. Furthermore, when cells were pretreated with sphingosine and then acutely treated with TPA, the acute TPA effect on kinase F_A/GSK-3 activity could be abolished by genistein or tyrosine phosphorylation, which could be blocked by genistein or tyrosine phosphatase, but could be reversed by orthovanadate. Taken together, the results demonstrate that TPA/sphingosine induce tyrosine phosphorylation and concurrent activation of kinase F_A/GSK-3 in a common signalling pathway. Since TPA and sphingosine are potent PKC modulators, the results further suggest a potential role of PKC in modulating tyrosine phosphorylation/activation of kinase F_A/GSK-3. Kinetic studies on seven subtypes of PKC further demonstrate a specific involvement of PKC in this tyrosine phosphorylation/activation process. This provides a new mode of signal transduction between these two important serine/threonine kinases in cells.     

Hydrogen Peroxide-Induced Phospholipase D Activation in Rat Pheochromocytoma PC12 Cells: Possible Involvement of Ca2+-Dependent Protein Tyrosine Kinase

Abstract: The mechanism for hydrogen peroxide (H₂O₂)-induced phospholipase D (PLD) activation was investigated in [³H]palmitic acid-labeled PC12 cells. In the presence of butanol, H₂O₂ caused a great accumulation of [³H]phosphatidylbutanol in a concentration- or time-dependent manner. However, treatment with H₂O₂ of cell lysates exerted no effect on PLD activity. Treatment with H₂O₂ had only a marginal effect on phospholipase C (PLC) activation. A protein kinase C (PKC) inhibitor, Ro 31-8220, did not inhibit but rather slightly enhanced H₂O₂-induced PLD activity. Thus, H₂O₂-induced PLD activation is considered to be independent of the PLC-PKC pathway in PC12 cells. In contrast, pretreatment with tyrosine kinase inhibitor herbimycin A, genistein, or ST638 resulted in a concentration-dependent inhibition of H₂O₂-induced PLD activation. Western blot analysis revealed several apparent tyrosine-phosphorylated protein bands after the H₂O₂ treatment and tyrosine phosphorylation of these proteins was inhibited by these tyrosine kinase inhibitors. Moreover, depletion of extracellular Ca²⁺ abolished H₂O₂-induced PLD activation and protein tyrosine phosphorylation. Extracellular Ca²⁺ potentiated H₂O₂-induced PLD activation in a concentration-dependent manner. Taken together, these results suggest that a certain Ca²⁺-dependent protein tyrosine kinase(s) somehow participates in H₂O₂-induced PLD activation in PC12 cells.     

Regulation of phospholipase D by muscarinic receptors in rat submandibular ductal cells

The muscarinic agonist carbachol stimulated phospholipase D (PLD) in rat submandibular gland (RSMG) ductal cells in a time and concentration-dependent manner. This effect was inhibited by chelation of extracellular calcium with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). PLD could also be activated by epinephrine and AlF(4)(-), two polyphosphoinositide-specific phospholipase C (PPI-PLC) activators, and by the phorbol ester o-tetradecanoylphorbol 13-acetate (TPA) which activates protein kinase C (PKC). Ionomycin and thapsigargin only slightly increased PLD activity. Ortho-vanadate, a tyrosine phosphatase inhibitor, also stimulated PLD activity. Both carbachol and o-vanadate increased the formation of inositol phosphates and the tyrosine phosphorylation of at least two proteins (55-60 and 120 kDa). Calphostin C (a PKC inhibitor), U73122 (a PPI-PLC inhibitor) and genistein (a tyrosine kinase inhibitor) blocked the activation of PLD, of PLC and the phosphorylation of tyrosyl residues in response to carbachol and vanadate. Taken together, these results suggest that rat submandibular gland ductal cells express a calcium-dependent PLD activity. This enzyme is regulated by carbachol via a PLC-PKC-tyrosine kinase pathway.     

Implication of Ca2+-Dependent Protein Tyrosine Phosphorylation in Carbachol-Induced Phospholipase D Activation in Rat Pheochromocytoma PC12 Cells

Abstract: The mechanism for carbachol (CCh)-induced phospholipase D (PLD) activation was investigated in [³H]palmitic acid-labeled pheochromocytoma PC12 cells with respect to the involvement of protein tyrosine phosphorylation and Ca²⁺. PLD activity was assessed by measuring the formation of [³H]phosphatidylbutanol in the presence of 0.3% butanol. Pretreatment of cells with the tyrosine kinase inhibitors herbimycin A, genistein, and tyrphostin inhibited PLD activation by CCh. Western blot analysis revealed several apparent tyrosine-phosphorylated protein bands (111, 91, 84, 74, 65–70, 44, and 42 kDa) in PC12 cells treated with CCh. Phosphorylation of the 111-, 91-, 84-, and 65–70-kDa proteins peaked within 1 min, and their time-dependent changes seemingly correlated with that of PLD activation. Others (74, 44^MAPK, and 42^MAPK kDa) were phosphorylated rather slowly, and maximal tyrosine phosphorylation was observed at 2 min. Herbimycin A inhibited PLD activity and tyrosine phosphorylation of four proteins (111, 91, 84, and 65–70 kDa) in a preincubation time- and concentration-dependent fashion. In Ca²⁺-free buffer, CCh-induced [³H]phosphatidylbutanol formation and protein tyrosine phosphorylation were abolished. A Ca²⁺ ionophore, A23187, caused PLD activation and tyrosine phosphorylation of four proteins of 111, 91, 84, and 65–70 kDa only in the presence of extracellular Ca²⁺. Extracellular Ca²⁺ dependency for CCh-induced PLD activation was well correlated with that for tyrosine phosphorylation of the four proteins listed above, especially the 111-kDa protein. These results suggest that Ca²⁺-dependent protein tyrosine phosphorylation is closely implicated in CCh-induced PLD activation in PC12 cells.     

Glutamate-induced protein phosphorylation in cerebellar granule cells: Role of protein kinase C

Protein phosphorylation in response to toxic doses of glutamate has been investigated in cerebellar granule cells.³²P-labelled cells have been stimulated with 100 M glutamate for up to 20 min and analysed by one and two dimensional gel electrophoresis. A progressive incorporation of label is observed in two molecular species of about 80 and 43 kDa (PP80 and PP43) and acidic isoelectric point. Glutamate-stimulated phosphorylation is greatly reduced by antagonists of NMDA and non-NMDA glutamate receptors. The effect of glutamate is mimicked by phorbol esters and is markedly reduced by inhibitors of protein kinase C (PKC) such as staurosporine and calphostin C. PP80 has been identified by Western blot analysis as the PKC substrate MARCKS (myristoylated alanine-rich C kinase substrate), while antibody to GAP-43 (growth associated protein-43), the nervous tissue-specific substrate of PKC, failed to recognize PP43. Our results suggest that PKC is responsible for the early phosphorylative events induced by toxic doses of glutamate in cerebellar granule cells.Abbreviations (NMDA) N-methyl-D-aspartate - (PKC) protein kinase C - (EAA) excitatory aminoacids - (GAMSA) -D-glutamylaminomethylsulfonate - (MK801) (+)-10,11-dihydro-5-methyl-5-H-dibenzo-(a,d)-cyclohepten-5,10imine - (TPA) phorbol 12-myristate 13-acetate - (MARCKS) myristoylated alanine-rich C kinase substrate - (GAP-43) growth-associated protein-43 - (SDS) sodium dodecyl sulfate - (PAGE) polyacrylamide gel electrophoresis - (H7) 1-(5-isoquinolinesulfonyl)-2-methylpiperazine - (DIV) days in vitro     

Regulation of acetylcholine-induced phosphorylation of PLD1 in porcine tracheal smooth muscle

The muscarinic agonist, acetylcholine (ACh), stimulates phospholipase D (PLD) activity in tracheal smooth muscle cells. Direct activation of protein kinase C (PKC) by phorbol-12-myristate-13-acetate (PMA) also stimulates PLD in this tissue. Activation of ACh-induced PLD was inhibited by the tyrosine kinase inhibitor genistein in a concentration-dependent manner. Presently known isoforms of PLD, PLD1 and PLD2, were identified in tracheal smooth muscle and their activation-induced phosphorylation status studied. Both ACh and PMA increased phosphorylation of PLD1 that was significantly blocked by genistein or the PKC inhibitor calphostin C. PLD2 phosphorylation was not detected in the present experiments. Western blots probed with an anti-phosphotyrosine antibody indicate that PLD1 in this tissue is phosphorylated on tyrosine residues after ACh or PMA stimulation. Tyrosine phosphorylation of PLD1 was blocked by genistein and calphostin C. No tyrosine residues were phosphorylated on PLD2. Taken together, these results demonstrate that porcine tracheal smooth muscle cells express both isoforms PLD1 and PLD2. However, on muscarinic activation only PLD1 in this tissue is phosphorylated by PKC via a tyrosine-kinase-dependent pathway.     

The Naturally Occurring PKC Inhibitor Sphingosine and Tumor Promoter Phorbol Ester Potentially Induce Tyrosine Phosphorylation/Activation of Oncogenic Proline-Directed Protein Kinase FA/GSK-3α in a Common Signalling Pathway

When serum-starved A431 cells were treated with 200 nM phorbol ester TPA for 15 min, the cellular activity of protein kinase F_A/glycogen synthase kinase-3α (kinase F_A/GSK-3α) could be decreased to ~25% of control. Conversely, when treated with 1 μM TPA for 24 hr, the activity could be reversibly increased to ~200% of Control. The naturally occurring protein kinase C (PKC) inhibitor sphingosine at a concentration of 27 μM could also induce activation of kinase F_A/GSK-3α to ~200% of control within 60 min. Further, when cells were chronically treated with 1 μM TPA for 24 hr and then with 27 μM sphingosine for 60 min, the activity of kinase F_A/GSK-3α could only be increased to ~200% of control. Furthermore, when cells were pretreated with sphingosine and then acutely treated with TPA, the acute TPA effect on kinase F_A/GSK-3α activity could be abolished by genistein or tyrosine phosphorylation, which could be blocked by genistein or tyrosine phosphatase, but could be reversed by orthovanadate. Taken together, the results demonstrate that TPA/sphingosine induce tyrosine phosphorylation and concurrent activation of kinase F_A/GSK-3α in a common signalling pathway. Since TPA and sphingosine are potent PKC modulators, the results further suggest a potential role of PKC in modulating tyrosine phosphorylation/activation of kinase F_A/GSK-3α. Kinetic studies on seven subtypes of PKC further demonstrate a specific involvement of PKC∈ in this tyrosine phosphorylation/activation process. This provides a new mode of signal transduction between these two important serine/threonine kinases in cells.     

Protein kinase C-alpha produces reciprocal effects on the phorbol ester stimulated tyrosine phosphorylation of a 50 kDa kinase in Jurkat cells.

A detergent extract isolated from the enriched fraction of integral membrane proteins of Jurkat cells showed an enhanced tyrosine phosphate level when phosphorylated in the presence of phorbol 12-myristate 13-acetate (TPA) and phorbol 12,13-dibutyrate (PDBu). The enhanced tyrosine phosphorylation was observed when the reaction time exceeded 6 min; at shorter incubation times, however, TPA inhibited tyrosine phosphorylation. When the reaction proceeded for a constant time period longer than 6 min and phorbol esters were added at different times after the start of the reaction, two phases of an enhanced tyrosine phosphorylation of a 50 kDa protein were observed. An increased phosphorylation of the 50 kDa protein was correlated with an enhanced phosphorylation of poly(Glu4,Tyr1). The two phases of enhanced phosphorylation differed in their TPA and PDBu requirement and in the proteins that were tyrosine phosphorylated. Studies with protein kinase C (PKC) inhibitors showed a negatively correlated effect on the enhanced tyrosine phosphorylation in phase I; tyrosine phosphorylation was further augmented. In phase II the regulation of tyrosine phosphorylation correlated with the efficiency of the PKC inhibitors on the alpha-isoform of PKC which was found in the cell extract. Separation of the proteins present in the investigated cell extract by gel filtration revealed a co-migration of the alpha-PKC and the 50 kDa protein. The metabolic labeling of intact Jurkat cells with 32Pi indicated that phorbol esters are also able to induce tyrosine phosphorylation of the 50 kDa protein underin vivo conditions. These data suggest an activation of two different tyrosine phosphorylation pathways by phorbol esters involving tyrosine phosphorylation/autophosphorylation of a 50 kDa kinase, as confirmed by 5'-p-fluorosulfonylbenzoyladenosine (FSBA) labeling, that are accurately regulated by alpha-PKC.     

Antagonistic effects of protein kinase C alpha and delta on both transformation and phospholipase D activity mediated by the epidermal growth factor receptor.

Downregulation of protein kinase C delta (PKC delta) by treatment with the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) transforms cells that overexpress the non-receptor class tyrosine kinase c-Src (Z. Lu et al., Mol. Cell. Biol. 17:3418-3428, 1997). We extended these studies to cells overexpressing a receptor class tyrosine kinase, the epidermal growth factor (EGF) receptor (EGFR cells); like c-Src, the EGF receptor is overexpressed in several human tumors. In contrast with expectations, downregulation of PKC isoforms with TPA did not transform the EGFR cells; however, treatment with EGF did transform these cells. Since TPA downregulates all phorbol ester-responsive PKC isoforms, we examined the effects of PKC delta- and PKC alpha-specific inhibitors and the expression of dominant negative mutants for both PKC delta and alpha. Consistent with a tumor-suppressing function for PKC delta, the PKC delta-specific inhibitor rottlerin and a dominant negative PKC delta mutant transformed the EGFR cells in the absence of EGF. In contrast, the PKC alpha-specific inhibitor Go6976 and expression of a dominant negative PKC alpha mutant blocked the transformed phenotype induced by both EGF and PKC delta inhibition. Interestingly, both rottlerin and EGF induced substantial increases in phospholipase D (PLD) activity, which is commonly elevated in response to mitogenic stimuli. The elevation of PLD activity in response to inhibiting PKC delta, like transformation, was dependent upon PKC alpha and restricted to the EGFR cells. These data demonstrate that PKC isoforms alpha and delta have antagonistic effects on both transformation and PLD activity and further support a tumor suppressor role for PKC delta that may be mediated by suppression of tyrosine kinase-dependent increases in PLD activity.     

Protein kinase C activates the renal apical membrane Na+/H+ exchanger

Summary Studies were performed on purified brush-border membranes from the kidney of the rabbit to examine the relation between protein kinase C and the Na⁺/H⁺ exchanger in these membranes. The brush-border membranes were transiently opened by exposure to hypotonic media and the membrane proteins phosphorylated by exposure to ATP and phorbol esters or partially purified protein kinase C. The membranes were resealed and the intravesicular space acidified by incubation in a sodium-free isotonic solution (pH 5.5). The rate of uptake of 1mm ²²Na⁺ (pH 7.5), with and without amiloride (1mm), was assayed and the proton gradient-stimulated, amiloride-inhibitable component of²²Na⁺ taken as a measure of the activity of the Na⁺/H⁺ exchanger. 12-0-tetradecanoyl phorbol-13-acetate (TPA) increased the amiloride-sensitive component of²²Na⁺ uptake TPA did not affect the amiloride-insensitive component of²²Na⁺ uptake or the equilibrium concentration of sodium. TPA also did not affect the rate of dissipation of the proton gradient in the absence of sodium or the rate of sodium-dependent or-independent uptake ofd-glucose. Other active phorbol esters stimulated the rate of Na⁺/H⁺ exchange, but phorbol esters of the 4 configuration did not. Incubation of the opened membranes in partially purified protein kinase C increased the rate of proton gradient-stimulated, amiloride-inhibitable sodium uptake. The stimulatory effect of TPA and protein kinase C was not additive. In the absence of ATP, neither TPA nor protein kinase C affected Na⁺/H⁺ exchange transport. To determine the membrane-bound protein substrates, parallel experiments were conducted with -[³²P] ATP in the phosphorylating solutions. The reaction was stopped by SDS and the phosphoproteins resolved by PAGE and autoradiography. TPA stimulation of protein kinase C resulted in phosphorylation of approximately 13 membrane-bound proteins ranging in apparent molecule from 15,000 to 140,000 daltons. These studies indicate that activation of endogenous renal brush-border protein kinase C by phorbol esters or exposure of these membranes to exogenous protein kinase C increases the rate of proton gradient-stimulated, amiloride-inhibitable sodium transport. Protein kinase C activation also results in phosphorylation of a finite number of membrane-bound proteins.     

Tumor promoter phorbol ester reversibly modulates tyrosine dephosphorylatioin/ inactivation of protein kinase FA/GSK-3α in A431 cells

The signal transducrion mechanism of protein kinase F_A/GSK-3α by tyrosine phosphorylation in A431 cells was investigated. Kinase F_A/GSK-3α was found to exist in a highly tyrosine-phosphorylated/activated state in resting cells but could be tyrosine-dephosphorylated and inactivated to ～60% of the control level when cells were acutely treated with 1 μM tumor phorbol ester (TPA) at 37^oC for 30 min, as demonstrated by metabolic ³²P-labeling the cells, followed by immunoprecipitation and two-dimensional phosphoamino acid analysis and by immunodetection in an antikinase F_A/GSK-3α immunoprecipitate kinase assay. Conversely, when cells were chronically treated with 1 μM TPA at 37°C for 24 h and processed under identical condetions, kinase F_A/GSK-3α was found to be rephosphorylated on tyrosine residue and reactivated to ～130% of the original control level. Taken together, the results provide initial evidence that the phosphotyrosine content and cellular activity of kinase F_A/GSK-3α can be modulated in a reversible manner by short-term and long-term exposure of A431 cells to TPA. Since acute exposure of cells to TPA causes up-regulation of cellular protein kinase C (PKC) activity and prolonged exposure to TPA causes down-regulation of PKC, the results further suggest that the TPA-mediated modulation of PKC may play a role in the regulation of tyrosine phosphorylation and concurrent activation of kinase F_A/GSK-3α in cells, representing a new mode of signal transduction pathway for the regulation of this multisubstrate/multifunctional protein kinase in cells.     

Identification of an autoinhibitory domain in the insulin receptor tyrosine kinase

We have tested the hypothesis that activation of the insulin receptor tyrosine kinase is due to autophosphorylation of tyrosines 1146, 1150 and 1151 within a putative autoinhibitory domain. A synthetic peptide corresponding to residues 1134–1162, with tyrosines substituted by alanine or phenylalanine, of the insulin receptor subunit was tested for its inhibitory potency and specificity towards the tyrosine kinase activity. This synthetic peptide gave inhibition of the insulin receptor tyrosine kinase autophosphorylation and phosphorylation of the exogenous substrate poly(Glu, Tyr) with an approximate IC₅₀ of 100 M. Inhibition appeared to be independent of the concentrations of insulin or the substrate poly(Glu, Tyr) but was decreased by increasing concentrations of ATP. This same peptide also inhibited the EGF receptor tyrosine kinase but not a serine/threonine protein kinase. These results are consistent with the hypothesis that this autophosphorylation domain contains an autoinhibitory sequence. (Mol Cell Biochem120: 103–110, 1993)Abbreviations IR Insulin Receptor - SDS/PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis - CaM Calmodulin - HEPES 4-(2-Hydroxyethyl)-Piperazineethane-Sulfonic Acid - DMEM Dulbecco's Modified Eagle' Medium - PMSF Phenylmethyl-Sulfonyl Fluoride - HPLC High Performance Liquid Chromatography - PKC Protein Kinase C - PKI Inhibitory Peptide for cAMP-Kinase - CaMK II Ca²⁺/Calmodulin-Dependent Protein Kinase II - CaN A A Subunit of Calcineurin     

Angiotensin II stimulates renal proximal tubule Na-ATPase activity through the activation of protein kinase C

Recently, our group described an AT₁-mediated direct stimulatory effect of angiotensin II (Ang II) on the Na⁺-ATPase activity of proximal tubules basolateral membranes (BLM) [Am. J. Physiol. 248 (1985) F621]. Data in the present report suggest the participation of a protein kinase C (PKC) in the molecular mechanism of Ang II-mediated stimulation of the Na⁺-ATPase activity due to the following observations: (i) the stimulation of protein phosphorylation in BLM, induced by Ang II, is mimicked by the PKC activator TPA, and is completely reversed by the specific PKC inhibitor, calphostin C; (ii) the Na⁺-ATPase activity is stimulated by Ang II and TPA in the same magnitude, being these effects abolished by the use of the PKC inhibitors, calphostin C and sphingosine; (iii) the Na⁺-ATPase activity is activated by catalytic subunit of PKC (PKC-M), in a similar and nonadditive manner to Ang II; and (iv) Ang II stimulates the phosphorylation of MARCKS, a specific substrate for PKC.     

PTTH-stimulated ERK phosphorylation in prothoracic glands of the silkworm, Bombyx mori: Role of Ca/calmodulin and receptor tyrosine kinase

Our previous studies showed that the prothoracicotropic hormone (PTTH) stimulated extracellular signal-regulated kinase (ERK) phosphorylation in prothoracic glands of Bombyx mori both in vitro and in vivo. In the present study, the signaling pathway by which PTTH activates ERK phosphorylation was further investigated using PTTH, second messenger analogs, and various inhibitors. ERK phosphorylation induced by PTTH was partially reduced in Ca²⁺-free medium. The calmodulin antagonist, calmidazolium, partially inhibited both PTTH-stimulated ERK phosphorylation and ecdysteroidogenesis, indicating the involvement of calmodulin. When the prothoracic glands were treated with agents that directly elevate the intracellular Ca²⁺ concentration [either A23187, thapsigargin, or the protein kinase C (PKC) activator, phorbol 12-myristate acetate (PMA)], a great increase in ERK phosphorylation was observed. In addition, it was found that PTTH-stimulated ecdysteroidogenesis was greatly attenuated by treatment with PKC inhibitors (either calphostin C or chelerythrine C). However, PTTH-stimulated ERK phosphorylation was not attenuated by the above PKC inhibitors, indicating that PKC is not involved in PTTH-stimulated ERK phosphorylation. A potent and specific inhibitor of insulin receptor tyrosine kinase, HNMPA-(AM)₃, greatly inhibited the ability of PTTH to activate ERK phosphorylation and stimulate ecdysteroidogenesis. However, genistein, another tyrosine kinase inhibitor, did not inhibit PTTH-stimulated ERK phosphorylation, although it did markedly attenuate the ability of A23187 to activate ERK phosphorylation. From these results, it is suggested that PTTH-stimulated ERK phosphorylation is only partially Ca²⁺- and calmodulin-dependent and that HNMPA-(AM)₃-sensitive receptor tyrosine kinase is involved in activation of ERK phosphorylation by PTTH.