Identification of tyrosines in the putative regulatory site of the Ca2+ channel TRPV6

In HEK293 cells, transfected with the Ca2+ channel protein TRPV6, Ca2+ influx is increased and TRPV6 is tyrosine phosphorylated following addition of the tyrosine phosphatase inhibitor N,N-dimethyl-hydroxamido hydroxovanadate to cells. This effect of DMHV is enhanced by co-transfection of cells with the tyrosine kinase Src and the tyrosine phosphatase 1B. It is abolished when cells had been treated with PP1, an inhibitor of Src family tyrosine kinases. PTP1B interacts with the N-terminal domain of TRPV6 within a region of amino acids 1-191 as shown by co-immunoprecipitation, bimolecular fluorescence complementation and the yeast 2-hybrid system. Point mutation of both tyrosines 161 and 162 in the TRPV6 protein abolishes the DMHV-effect on Ca2+ influx and tyrosine phosphorylation by Src. Single mutations of Y161 or Y162 shows that each of both tyrosines alone is sufficient for the DMHV-effect. We conclude that phosphorylation/dephosphorylation of tyrosines in position 161 and 162 is essential for regulation of Ca2+ influx through TRPV6 Ca2+ channels in HEK293 cells.     

The signalling cascade of fertilization

The early event of fertilization-induced egg activation is a mobilization of intracellular Ca2+ that originates from the sperm entry point and spreads through the entire egg cytoplasm. Recently, this process has been established to require the sequential activation of Src family kinases, phospholipase C gamma, and inositoltrisphosphate receptor of endoplasmic reticulum. This review summarizes recent findings concerning the signalling pathway of fertilization from sperm-egg interaction to the Ca2+ release with emphasis on the role of tyrosine kinases in the egg activation.     

Involvement of JAK2 and Src kinase tyrosine phosphorylation in human growth hormone-stimulated increases in cytosolic free Ca2+ and insulin secretion

We previously reported that human growth hormone (hGH) increases cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) and proliferation in pancreatic -cells (Sjöholm Å, Zhang Q, Welsh N, Hansson A, Larsson O, Tally M, and Berggren PO. J Biol Chem 275: 21033–21040, 2000) and that the hGH-induced rise in [Ca²⁺]_i involves Ca²⁺-induced Ca²⁺ release facilitated by tyrosine phosphorylation of ryanodine receptors (Zhang Q, Kohler M, Yang SN, Zhang F, Larsson O, and Berggren PO. Mol Endocrinol 18: 1658–1669, 2004). Here we investigated the tyrosine kinases that convey the hGH-induced rise in [Ca²⁺]_i and insulin release in BRIN-BD11 -cells. hGH caused tyrosine phosphorylation of Janus kinase (JAK)2 and c-Src, events inhibited by the JAK2 inhibitor AG490 or the Src kinase inhibitor PP2. Although hGH-stimulated rises in [Ca²⁺]_i and insulin secretion were completely abolished by AG490 and JAK2 inhibitor II, the inhibitors had no effect on insulin secretion stimulated by a high K⁺ concentration. Similarly, Src kinase inhibitor-1 and PP2, but not its inactive analog PP3, suppressed [Ca²⁺]_i elevation and completely abolished insulin secretion stimulated by hGH but did not affect responses to K⁺. Ovine prolactin increased [Ca²⁺]_i and insulin secretion to a similar extent as hGH, effects prevented by the JAK2 and Src kinase inhibitors. In contrast, bovine GH evoked a rise in [Ca²⁺]_i but did not stimulate insulin secretion. Neither JAK2 nor Src kinase inhibitors influenced the effect of bovine GH on [Ca²⁺]_i. Our study indicates that hGH stimulates rise in [Ca²⁺]_i and insulin secretion mainly through activation of the prolactin receptor and JAK2 and Src kinases in rat insulin-secreting cells. c-Src; growth hormone receptor; prolactin receptor; Ca²⁺-induced Ca²⁺ release     

Angiotensin II induces focal adhesion kinase/paxillin phosphorylation and cell migration in human umbilical vein endothelial cells

In the present study, we demonstrated that Ang II provokes a transitory enhancement of focal adhesion kinase (FAK) and paxillin phosphorylation in human umbilical endothelial cells (HUVEC). Moreover, Ang II induces a time- and dose-dependent augmentation in cell migration, but does not affect HUVEC proliferation. The effect of Ang II on FAK and paxillin phosphorylation was markedly attenuated in cells pretreated with wortmannin and LY294002, indicating that phosphoinositide 3-kinase (PI3K) plays an important role in regulating FAK activation. Similar results were observed when HUVEC were pretreated with genistein, a non-selective tyrosine kinases inhibitor, or with the specific inhibitor PP2 for Src family kinases, demonstrating the involvement of protein tyrosine kinases, and particularly Src family of tyrosine kinases, in the downstream signalling pathway of Ang II receptors. Furthermore, FAK and paxillin phosphorylation was markedly blocked after treatment of HUVEC with AG1478, a selective inhibitor of epidermal growth factor receptor (EGFR) phosphorylation. Pretreatment of cells with inhibitors of PI3K, Src family tyrosine kinases, and EGFR also decreased HUVEC migration. In conclusion, these results suggest that Ang II mediates an increase in FAK and paxillin phosphorylation and induces HUVEC migration through signal transduction pathways dependent on PI3K and Src tyrosine kinase activation and EGFR transactivation.     

Muscarinic activation of mitogen-activated protein kinase in rat thyroid epithelial cells

Carbachol (Cch), a muscarinic acetylcholine receptors (mAChR) agonist, produces time- and dose-dependent increases in mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) phosphorylation in nondifferentiated Fischer rat thyroid (FRT) epithelial cells. Cells pretreatment with the selective phospholipase C inhibitor U73122 resulted in a decrease of Cch-stimulated ERK1/2 phosphorylation. These data indicated that the effect of mAChR on ERK activation could be mediated through agonist-induced Ca(2+) mobilization or PKC activation. Phosphorylation of ERK1/2 was mimicked by the protein kinase C (PKC) activator phorbol 12-myristate acetate (PMA), but was not altered either by PKC inhibitor GF109203X or by down-regulation of PKC. Phosphorylation of ERK1/2 was elevated by a direct [Ca(2+)](i) increase caused by thapsigargin or ionophore. Additionally, Cch-induced ERK1/2 phosphorylation was reduced after either inhibition of Ca(2+) influx or intracellular Ca(2+) release. Nevertheless, Cch-mediated ERK1/2 activation was genistein sensitive, indicating the involvement of protein tyrosine kinases on the downstream signalling of mAChR. Pretreatment of the cells with PP2 markedly decreased Cch-induced ERK1/2 phosphorylation, suggesting a role of Src family of tyrosine kinases in the signal transduction pathway involved in ERK1/2 activation by mAChR. To test the biological consequences of ERK activation, we examined the effect of mAChR on cell functions. Cch stimulation of FRT cells did not affect cell proliferation, but increased protein synthesis. This effect was significantly attenuated by PD98059, a selective inhibitor of mitogen-activated protein kinase kinase (MAPKK/MEK). This study demonstrated that muscarinic receptor-mediated increase in the ERK1/2 phosphorylation was dependent on [Ca(2+)](i) but independent of PKC and was mediated by the Src family of tyrosine kinases. Our results also supported the idea that the protein synthesis stimulated by mAChR in polarized FRT epithelial cells was regulated by the ERK1/2 phosphorylation pathway.     

Src tyrosine kinase regulates insulin-induced activation of protein kinase C (PKC) delta in skeletal muscle

Insulin stimulation of skeletal muscle results in rapid activation of protein kinase Cdelta (PKCdelta), which is associated with its tyrosine phosphorylation and physical association with insulin receptor (IR). The mechanisms underlying tyrosine phosphorylation of PKCdelta have not been determined. In this study, we investigated the possibility that the Src family of nonreceptor tyrosine kinases may be involved upstream insulin signaling. Studies were done on differentiated rat skeletal myotubes in primary culture. Insulin caused an immediate stimulation of Src and induced its physical association with both IR and PKCdelta. Inhibition of Src by treatment with the Src family inhibitor PP2 reduced insulin-stimulated Src-PKCdelta association, PKCdelta tyrosine phosphorylation and PKCdelta activation. PP2 inhibition of Src also decreased insulin-induced IR tyrosine phosphorylation, IR-PKCdelta association and association of Src with both PKCdelta and IR. Finally, inhibition of Src decreased insulin-induced glucose uptake. We conclude that insulin activates Src tyrosine kinase, which regulates PKCdelta activity. Thus, Src tyrosine kinase may play an important role in insulin-induced tyrosine phosphorylation of both IR and PKCdelta. Moreover, both Src and PKCdelta appear to be involved in IR activation and subsequent downstream signaling.     

Regulation of the type 1 inositol 1,4,5-trisphosphate receptor by phosphorylation at tyrosine 353

The inositol 1,4,5-trisphosphate receptor (IP3R) plays an essential role in Ca2+ signaling during lymphocyte activation. Engagement of the T cell or B cell receptor by antigen initiates a signal transduction cascade that leads to tyrosine phosphorylation of IP3R by Src family nonreceptor protein tyrosine kinases, including Fyn. However, the effect of tyrosine phosphorylation on the IP3R and subsequent Ca2+ release is poorly understood. We have identified tyrosine 353 (Tyr353) in the IP3-binding domain of type 1 IP3R (IP3R1) as a phosphorylation site for Fyn both in vitro and in vivo. We have developed a phosphoepitope-specific antibody and shown that IP3R1-Y353 becomes phosphorylated during T cell and B cell activation. Furthermore, tyrosine phosphorylation of IP3R1 increased IP3 binding at low IP3 concentrations (<10 nm). Using wild-type IP3R1 or an IP3R1-Y353F mutant that cannot be tyrosine phosphorylated at Tyr353 or expressed in IP3R-deficient DT40 B cells, we demonstrated that tyrosine phosphorylation of Tyr353 permits prolonged intracellular Ca2+ release during B cell activation. Taken together, these data suggest that one function of tyrosine phosphorylation of IP3R1-Y353 is to enhance Ca2+ signaling in lymphocytes by increasing the sensitivity of IP3R1 to activation by low levels of IP3.     

Complexes of gamma-tubulin with nonreceptor protein tyrosine kinases Src and Fyn in differentiating P19 embryonal carcinoma cells

Nonreceptor protein tyrosine kinases of the Src family have been shown to play an important role in signal transduction as well as in regulation of microtubule protein interactions. Here we show that gamma-tubulin (gamma-Tb) in P19 embryonal carcinoma cells undergoing neuronal differentiation is phosphorylated and forms complexes with protein tyrosine kinases of the Src family, Src and Fyn. Elevated expression of both kinases during differentiation corresponded with increased level of proteins phosphorylated on tyrosine. Immunoprecipitation experiments with antibodies against Src, Fyn, gamma-tubulin, and with anti-phosphotyrosine antibody revealed that gamma-tubulin appeared in complexes with these kinases. In vitro kinase assays showed tyrosine phosphorylation of proteins in gamma-tubulin complexes isolated from differentiated cells. Pretreatment of cells with Src family selective tyrosine kinase inhibitor PP2 reduced the amount of phosphorylated gamma-tubulin in the complexes. Binding experiments with recombinant SH2 and SH3 domains of Src and Fyn kinases revealed that protein complexes containing gamma-tubulin bound to SH2 domains and that these interactions were of SH2-phosphotyrosine type. The combined data suggest that Src family kinases might have an important role in the regulation of gamma-tubulin interaction with tubulin dimers or other proteins during neurogenesis.     

Inhibiting Src family tyrosine kinase activity blocks glutamate signalling to ERK1/2 and Akt/PKB but not JNK in cultured striatal neurones

Glutamate receptor activation of mitogen-activated protein (MAP) kinase signalling cascades has been implicated in diverse neuronal functions such as synaptic plasticity, development and excitotoxicity. We have previously shown that Ca2+-influx through NMDA receptors in cultured striatal neurones mediates the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt/protein kinase B (PKB) through a phosphatidylinositol 3-kinase (PI 3-kinase)-dependent pathway. Exposing neurones to the Src family tyrosine kinase inhibitor PP2, but not the inactive analogue PP3, inhibited NMDA receptor-induced phosphorylation of ERK1/2 and Akt/PKB in a concentration-dependent manner, and reduced cAMP response element-binding protein (CREB) phosphorylation. To establish a link between Src family tyrosine kinase-mediated phosphorylation and PI 3-kinase signalling, affinity precipitation experiments were performed with the SH2 domains of the PI 3-kinase regulatory subunit p85. This revealed a Src-dependent phosphorylation of a focal adhesion kinase (FAK)-p85 complex on glutamate stimulation. Demonstrating that PI3-kinase is not ubiquitously involved in NMDA receptor signal transduction, the PI 3-kinase inhibitors wortmannin and LY294002 did not prevent NMDA receptor Ca2+-dependent phosphorylation of c-Jun N-terminal kinase 1/2 (JNK1/2). Further, inhibiting Src family kinases increased NMDA receptor-dependent JNK1/2 phosphorylation, suggesting that Src family kinase-dependent cascades may physiologically limit signalling to JNK. These results demonstrate that Src family tyrosine kinases and PI3-kinase are pivotal regulators of NMDA receptor signalling to ERK/Akt and JNK in striatal neurones.     

Wheat germ agglutinin-induced platelet activation via platelet endothelial cell adhesion molecule-1: involvement of rapid phospholipase C gamma 2 activation by Src family kinases.

Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) is a 130K transmembrane glycoprotein that belongs to the immunoglobulin gene superfamily and is expressed on the surface of hematological or vascular cells, including platelets and endothelial cells. Although the importance of this adhesion molecule in various cell-cell interactions is established, its function in platelets remains ill-defined. In the process of clarifying the mechanism by which the lectin wheat germ agglutinin (WGA) activates platelets, we unexpectedly discovered that PECAM-1 is involved in signal transduction pathways elicited by this N-acetyl-D-glucosamine (NAGlu)-reactive lectin. WGA, which is a very potent platelet stimulator, elicited a rapid surge in Syk and phospholipase C (PLC)-gamma 2 tyrosine phosphorylation and the resultant intracellular Ca(2+) mobilization; collagen, as reported, induced these responses, but in a much slower and weaker manner. WGA strongly induced tyrosine phosphorylation of a 130-140K protein, which was confirmed to be PECAM-1 by immunoprecipitation and immunodepletion studies. WGA-induced PECAM-1 tyrosine phosphorylation occurred rapidly, strongly and in a manner independent of platelet aggregation or cell-cell contact; these characteristics of PECAM-1 phosphorylation were not mimicked at all by receptor-mediated platelet agonists. In addition, WGA was found to associate with PECAM-1 itself, and anti-PECAM-1 antibody, as well as NAGlu, specifically inhibited WGA-induced platelet aggregation. In PECAM-1 immunoprecipitates, Src family tyrosine kinases existed, and a kinase activity was detected, which increased upon WGA stimulation. Furthermore, the Src family kinase inhibitor PP2 inhibited WGA-induced platelet aggregation, Ca(2+) mobilization, and PLC-gamma 2 tyrosine phosphorylation. Finally, WGA induced PECAM-1 tyrosine phosphorylation and cytoskeletal reorganization in vascular endothelial cells. Our results suggest that (i) PECAM-1 is involved in WGA-induced platelet activation, (ii) PECAM-1 clustering by WGA activates unique and strong platelet signaling pathways, leading to a rapid PLC activation via Src family kinases, and (iii) WGA is a useful tool for elucidating PECAM-1-mediated signaling with wide implications not confined to platelets.     

Cell shrinkage regulates Src kinases and induces tyrosine phosphorylation of cortactin, independent of the osmotic regulation of Na+/H+ exchangers

The signaling pathways by which cell volume regulates ion transporters, e.g. Na+/H+ exchangers (NHEs), and affects cytoskeletal organization are poorly understood. We have previously shown that shrinkage induces tyrosine phosphorylation in CHO cells, predominantly in an 85-kDa band. To identify volume-sensitive kinases and their substrates, we investigated the effect of hypertonicity on members of the Src kinase family. Hyperosmolarity stimulated Fyn and inhibited Src. Fyn activation was also observed in nystatin-permeabilized cells, where shrinkage cannot induce intracellular alkalinization. In contrast, osmotic inhibition of Src was prevented by permeabilization or by inhibiting NHE-1. PP1, a selective Src family inhibitor, strongly reduced the hypertonicity-induced tyrosine phosphorylation. We identified one of the major targets of the osmotic stress-elicited phosphorylation as cortactin, an 85-kDa actin-binding protein and well known Src family substrate. Cortactin phosphorylation was triggered by shrinkage and not by changes in osmolarity or pHi and was abrogated by PP1. Hyperosmotic cortactin phosphorylation was reduced in Fyn-deficient fibroblasts but remained intact in Src-deficient fibroblasts. To address the potential role of the Src family in the osmotic regulation of NHEs, we used PP1. The drug affected neither the hyperosmotic stimulation of NHE-1 nor the inhibition of NHE-3. Thus, members of the Src family are volume-sensitive enzymes that may participate in the shrinkage-related reorganization of the cytoskeleton but are probably not responsible for the osmotic regulation of NHE.     

Antagonism of rat beta-cell voltage-dependent K+ currents by exendin 4 requires dual activation of the cAMP/protein kinase A and phosphatidylinositol 3-kinase signaling pathways

Antagonism of voltage-dependent K+ (Kv) currents in pancreatic beta-cells may contribute to the ability of glucagon-like peptide-1 (GLP-1) to stimulate insulin secretion. The mechanism and signaling pathway regulating these currents in rat beta-cells were investigated using the GLP-1 receptor agonist exendin 4. Inhibition of Kv currents resulted from a 20-mV leftward shift in the voltage dependence of steady-state inactivation. Blocking cAMP or protein kinase A (PKA) signaling (Rp-cAMP and H-89, respectively) prevented the inhibition of currents by exendin 4. However, direct activation of this pathway alone by intracellular dialysis of cAMP or the PKA catalytic subunit (cPKA) could not inhibit currents, implicating a role for alternative signaling pathways. A number of phosphorylation sites associated with phosphatidylinositol 3 (PI3)-kinase activation were up-regulated in GLP-1-treated MIN6 insulinoma cells, and the PI3 kinase inhibitor wortmannin could prevent antagonism of beta-cell currents by exendin 4. Antagonists of Src family kinases (PP1) and the epidermal growth factor (EGF) receptor (AG1478) also prevented current inhibition by exendin 4, demonstrating a role for Src kinase-mediated trans-activation of the EGF tyrosine kinase receptor. Accordingly, the EGF receptor agonist betacellulin could replicate the effects of exendin 4 in the presence of elevated intracellular cAMP. Downstream, the PKCzeta pseudosubstrate inhibitor could prevent current inhibition by exendin 4. Therefore, antagonism of beta-cell Kv currents by GLP-1 receptor activation requires both cAMP/PKA and PI3 kinase/PKCzeta signaling via trans-activation of the EGF receptor. This represents a novel dual pathway for the control of Kv currents by G protein-coupled receptors.     

The Src family kinases Hck and Fgr regulate neutrophil responses to N-formyl-methionyl-leucyl-phenylalanine

The chemotactic peptide formyl-methionyl-leucyl-phenilalanine (fMLP) triggers intracellular protein tyrosine phosphorylation leading to neutrophil activation. Deficiency of the Src family kinases Hck and Fgr have previously been found to regulate fMLP-induced degranulation. In this study, we further investigate fMLP signaling in hck-/-fgr-/- neutrophils and find that they fail to activate a respiratory burst and display reduced F-actin polymerization in response to fMLP. Additionally, albeit migration of both hck-/-fgr-/-mouse neutrophils and human neutrophils incubated with the Src family kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) through 3-microm pore size Transwells was normal, deficiency, or inhibition, of Src kinases resulted in a failure of neutrophils to migrate through 1-microm pore size Transwells. Among MAPKs, phosphorylation of ERK1/2 was not different, phosphorylation of p38 was only partially affected, and phosphorylation of JNK was markedly decreased in fMLP-stimulated hck-/-fgr-/- neutrophils and in human neutrophils incubated with PP2. An increase in intracellular Ca(2+) concentration and phosphorylation of Akt/PKB occurred normally in fMLP-stimulated hck-/-fgr-/- neutrophils, indicating that activation of both phosphoinositide-specific phospholipase C and PI3K is independent of Hck and Fgr. In contrast, phosphorylation of the Rho/Rac guanine nucleotide exchange factor Vav1 and the Rac target p21-activated kinases were markedly reduced in both hck-/-fgr-/- neutrophils and human neutrophils incubated with a PP2. Consistent with these findings, PP2 inhibited Rac2 activation in human neutrophils. We suggest that Hck and Fgr act within a signaling pathway triggered by fMLP receptors that involves Vav1 and p21-activated kinases, leading to respiratory burst and F-actin polymerization.     

Novel regulation of Na, K-ATPase by Src tyrosine kinases in cortical neurons

The Na+, K+-ATPase or Na+, K+-pump plays a critical role in ion homeostasis and many cellular events. The Na+, K+-pump activity is regulated by serine/threonine phosphorylation, the role of tyrosine kinases in the regulation, however, is obscure. We now present novel evidence showing that tyrosine phosphorylation activates the Na+, K+-pump in cortical neurons. The electrogenic activity of the Na+, K+-pump was measured using whole-cell voltage clamp. A tonic activity was revealed by an inward current induced by the specific inhibitor ouabain or strophanthidin; an outward current due to activation of the pump was triggered by raising extracellular K+. The inward and outward currents were attenuated by the tyrosine kinase inhibitor genistein, herbimycin A, or lavendustin A, while blocking tyrosine phosphatases increased the pump current. Down-regulation of the pump current was also seen with the Src inhibitor PP1 and intracellularly applied anti-Lyn or anti-Yes antibody. Consistently, intracellular application of Lyn kinase up-regulated the pump current. Immunoprecipitation and western blotting showed tyrosine phosphorylation and a direct interaction between Lyn and the alpha3 subunit of the Na+, K+-pump. The tyrosine phosphorylation of the alpha3 subunit was reduced by serum deprivation. These data suggest that the Na+, K+-ATPase activity in central neurons is regulated by specific Src tyrosine kinases via a protein-protein mechanism and may play a role in apoptosis.     

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.     

Tetracaine stimulates insulin secretion from the pancreatic beta-cell by release of intracellular calcium

The role of intracellular calcium stores in stimulus-secretion coupling in the pancreatic beta-cell is largely unknown. We report here that tetracaine stimulates insulin secretion from collagenase-isolated mouse islets of Langerhans in the absence of glucose or extracellular calcium. We also found that the anesthetic evokes a dose-dependent rise of the intracellular free-calcium concentration ([Ca2+]i) in cultured rat and mouse beta-cells. The tetracaine-specific [Ca2+]i rise also occurs in the absence of glucose, or in beta-cells depolarized by exposure to a Ca(2+)-deficient medium (< 1 microM) or elevated [K+]o. Furthermore, tetracaine (> or = 300 microM) depolarized the beta-cell membrane in mouse pancreatic islets, but inhibited Ca2+ entry through voltage-gated Ca2+ channels in HIT cells, an insulin-secreting cell line. From these data we conclude that tetracaine-enhancement of insulin release occurs by mechanisms that are independent of Ca2+ entry across the cell membrane. The tetracaine-induced [Ca2+]i rise in cultured rat beta-cells and insulin secretion from mouse islets is insensitive to dantrolene (20 microM), a drug that inhibits Ca2+ release evoked by cholinergic agonists in the pancreatic beta-cell, and thapsigargin (3 microM), a blocker of the endoplasmic reticulum (ER) Ca2+ pump. We conclude that the Ca2+ required for tetracaine-potentiated insulin secretion is released from intracellular Ca2+ stores other than the ER. Furthermore, tetracaine-induced Ca2+ release was unaffected by the mitochondrial electron transfer inhibitors NaN3 and rotenone. Taken together, these data show that a calcium source other than the ER and mitochondria can affect beta-cell insulin secretion.     

Src family tyrosine kinases differentially modulate exocytosis from rat brain nerve terminals

We have studied the role of src family tyrosine kinases in regulating synaptic transmitter release from rat brain synaptosomes by using two assays that measure different aspects of synaptic vesicle exocytosis: glutamate release (that directly measures exocytosis of vesicle contents) and release of FM 2-10 styryl dye (that is proportional to the time the synaptic vesicle is fused to the plasma membrane). Depolarisation was induced by KCl (30 mM) or 4-aminopyridine (4AP: 0.3mM) to induce release by full fusion (FF) exocytosis, or by 1 mM 4AP to induce release by both FF and kiss-and-run (KR)-like exocytosis. The src family selective inhibitor, PP1 (10 microM), increased KCl and 0.3 mM 4AP-evoked Ca2+ -dependent release of glutamate, but had little effect upon exocytosis evoked by 1mM 4AP. PP1 did not affect the release of FM 2-10 under any of the depolarisation conditions used. PP1 also had no effect on overall intracellular calcium levels, as measured by FURA2, suggesting that the effects of the inhibitor are downstream of calcium entry. At the same concentration the inactive analogue of this compound, PP3, had no effect on any measure. Immunoblotting with an antibody to phosphotyrosine revealed that phosphorylation of many synaptosomal proteins was reduced by PP1. The immunoreactivity of three protein bands increased upon depolarisation and this increase was blocked by PP1. Phosphorylation of src at tyrosine-416 was reduced by PP1 but changes in its phosphorylation did not correlate with the effects of PP1 on release. These results suggest one or more members of the src family of tyrosine kinases is a negative regulator of the KR mode of exocytosis in synaptosomes, perhaps by tonically inhibiting KR under normal stimulation conditions.     

Mildly oxidized low density lipoprotein rapidly stimulates via activation of the lysophosphatidic acid receptor Src family and Syk tyrosine kinases and Ca2+ influx in human platelets

In contrast to native low density lipoprotein (LDL), mildly oxidized LDL (mox-LDL) induced platelet shape change and stimulated during shape change the tyrosine phosphorylation of specific proteins including Syk; the translocation of Src, Fyn, and Syk to the cytoskeleton; and the increase of cytosolic Ca(2+) due to mainly Ca(2+) entry. The stimulation of these early signal pathways by mox-LDL was inhibited by desensitization of the lysophosphatidic acid (LPA) receptor and specific LPA receptor antagonists, was independent of the alpha(IIb)beta(3)-integrin, and was mimicked by LPA. Stimulation of tyrosine phosphorylation and Syk activation were independent of the increase of cytosolic Ca(2+) and were suppressed by genistein and two specific inhibitors of the Src family tyrosine kinases, PP1 and PD173956. In contrast to PP1 and PD 173956, genistein prevented shape change by mox-LDL. The results indicate that mox-LDL, through activation of the LPA receptor, stimulates two separate early signal pathways, (a) Src family and Syk tyrosine kinases, and (b) Ca(2+) entry. The activation of these early signaling pathways by mox-LDL probably plays a role in platelet responses subsequent to shape change. The inhibition of mox-LDL-induced platelet activation by LPA receptor antagonists or dietary isoflavonoids such as genistein could have implications in the prevention and therapy of cardiovascular diseases.     

Tyrosine 402 Phosphorylation of Pyk2 Is Involved in Ionomycin-Induced Neurotransmitter Release

Protein tyrosine kinases, which are highly expressed in the central nervous system, are implicated in many neural processes. However, the relationship between protein tyrosine kinases and neurotransmitter release remains unknown. In this study, we found that ionomycin, a Ca²⁺ ionophore, concurrently induced asynchronous neurotransmitter release and phosphorylation of a non-receptor protein tyrosine kinase, proline-rich tyrosine kinase 2 (Pyk2), in clonal rat pheochromocytoma PC12 cells and cerebellar granule cells, whereas introduction of Pyk2 siRNA dramatically suppressed ionomycin-induced neurotransmitter release. Further study indicated that Tyr-402 (Y402) in Pyk2, instead of other tyrosine sites, underwent rapid phosphorylation after ionomycin induction in 1 min to 2 min. We demonstrated that the mutant of Pyk2 Y402 could abolish ionomycin-induced dopamine (DA) release by transfecting cells with recombinant Pyk2 and its mutants (Y402F, Y579F, Y580F, and Y881F). In addition, Src inhibition could prolong phosphorylation of Pyk2 Y402 and increase DA release. These findings suggested that Pyk2 was involved in ionomycin-induced neurotransmitter release through phosphorylation of Y402.