Inhibitory role of Src family tyrosine kinases on Ca2+-dependent insulin release

Both neurotransmitter release and insulin secretion occur via regulated exocytosis and share a variety of similar regulatory mechanisms. It has been suggested that Src family tyrosine kinases inhibit neurotransmitter release from neuronal cells (H. Ohnishi, S. Yamamori, K. Ono, K. Aoyagi, S. Kondo, and M. Takahashi. Proc Natl Acad Sci USA 98: 10930–10935, 2001). Thus the potential role of Src family kinases in the regulation of insulin secretion was investigated in this study. Two structurally different inhibitors of Src family kinases, SU-6656 and PP2, but not the inactive compound, PP3, enhanced Ca2+-induced insulin secretion in both rat pancreatic islets and INS-1 cells in a concentration-dependent and time-dependent manner. Furthermore, Src family kinase-mediated insulin secretion appears to be dependent on elevated intracellular Ca2+ and independent of glucose metabolism, the ATP-dependent K+ channel, adenylyl cyclase, classical PKC isoforms, extracellular signal-regulated kinase 1/2, and insulin synthesis. The sites of action for Src family kinases seem to be distal to the elevation of intracellular Ca2+ level. These results indicate that one or more Src family tyrosine kinases exert a tonic inhibitory role on Ca2+-dependent insulin secretion.

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Src family kinases mediate neutrophil adhesion to adherent platelets

Blood ◽

10.1182/blood-2006-06-029082 ◽

2006 ◽

Vol 109 (6) ◽

pp. 2461-2469 ◽

Cited By ~ 84

Author(s):

Virgilio Evangelista ◽

Zehra Pamuklar ◽

Antonio Piccoli ◽

Stefano Manarini ◽

Giuseppe Dell'Elba ◽

...

Keyword(s):

Tyrosine Kinases ◽

Src Family Kinases ◽

Β2 Integrin ◽

Contact Sites ◽

Downstream Effector ◽

Activated Platelets ◽

Genetic Deletion ◽

Src Family Tyrosine Kinases

Abstract Polymorphonuclear leukocyte (PMN)–platelet interactions at sites of vascular damage contribute to local and systemic inflammation. We sought to determine the role of “outside-in” signaling by Src-family tyrosine kinases (SFKs) in the regulation of αMβ2-integrin–dependent PMN recruitment by activated platelets under (patho)physiologic conditions. Activation-dependent epitopes in β2 integrin were exposed at the contact sites between PMNs and platelets and were abolished by SFK inhibitors. PMNs from αMβ2−/−, hck−/−fgr−/−, and hck−/−fgr−/−lyn−/− mice had an impaired capacity to adhere with activated platelets in suspension. Phosphorylation of Pyk2 accompanied PMN adhesion to platelets and was blocked by inhibition as well as by genetic deletion of αMβ2 integrin and SFKs. A Pyk2 inhibitor reduced platelet-PMN adhesion, indicating that Pyk2 may be a downstream effector of SFKs. Analysis of PMN-platelet interactions under flow revealed that SFK signaling was required for αMβ2-mediated shear-resistant adhesion of PMNs to adherent platelets, but was dispensable for P-selectin–PSGL-1–mediated recruitment and rolling. Finally, SFK activity was required to support PMN accumulation along adherent platelets at the site of vascular injury, in vivo. These results definitely establish a role for SFKs in PMN recruitment by activated platelets and suggest novel targets to disrupt the pathophysiologic consequences of platelet-leukocyte interactions in vascular disease.

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Species-Specific Interactions of Src Family Tyrosine Kinases Regulate Chlamydia Intracellular Growth and Trafficking

mBio ◽

10.1128/mbio.00082-11 ◽

2011 ◽

Vol 2 (3) ◽

Cited By ~ 2

Author(s):

Cherilyn A. Elwell ◽

Arlinet Kierbel ◽

Joanne N. Engel

Keyword(s):

Tyrosine Kinases ◽

Bacterial Pathogenesis ◽

Src Family Kinases ◽

Intracellular Pathogens ◽

Specific Interactions ◽

Intracellular Growth ◽

Cellular Processes ◽

Src Family Tyrosine Kinases ◽

Species Specific

ABSTRACT Src family kinases (SFKs) regulate key cellular processes and are emerging as important targets for intracellular pathogens. In this commentary, we briefly review the role of SFKs in bacterial pathogenesis and highlight new work on the role of SFKs during the intracellular cycle of Chlamydia species.

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Cross talk between serine/threonine and tyrosine kinases regulates ADP-induced thromboxane generation in platelets

Thrombosis and Haemostasis ◽

10.1160/th14-09-0775 ◽

2015 ◽

Vol 114 (09) ◽

pp. 558-568 ◽

Cited By ~ 5

Author(s):

Dheeraj Bhavanasi ◽

Rachit Badolia ◽

Bhanu Kanth Manne ◽

Sumalaxmi Janapati ◽

Carol Dangelmaier ◽

...

Keyword(s):

Tyrosine Kinases ◽

Functional Role ◽

Human Platelets ◽

Src Family Kinases ◽

Dependent Manner ◽

Kinase C ◽

Pkc Isoforms ◽

First Time ◽

Stimulation Of

SummaryADP-induced thromboxane generation depends on Src family kinases (SFKs) and is enhanced with pan-protein kinase C (PKC) inhibitors, but it is not clear how these two events are linked. The aim of the current study is to investigate the role of Y311 phosphorylated PKCδ in regulating ADP-induced platelet activation. In the current study, we employed various inhibitors and murine platelets from mice deficient in specific molecules to evaluate the role of PKCδ in ADP-induced platelet responses. We show that, upon stimulation of platelets with 2MeSADP, Y311 on PKCδ is phosphorylated in a P2Y1/Gq and Lyn-dependent manner. By using PKCδ and Lyn knockout murine platelets, we also show that tyrosine phosphorylated PKCδ plays a functional role in mediating 2MeSADP-induced thromboxane generation. 2MeSADP-induced PKCδ Y311 phosphorylation and thromboxane generation were potentiated in human platelets pre-treated with either a pan-PKC inhibitor, GF109203X or a PKC α/β inhibitor and in PKC α or β knockout murine platelets compared to controls. Furthermore, we show that PKC α/β inhibition potentiates the activity of SFK, which further hyper-phosphorylates PKCδ and potentiates thromboxane generation. These results show for the first time that tyrosine phosphorylated PKCδ regulates ADP-induced thromboxane generation independent of its catalytic activity and that classical PKC isoforms α/β regulate the tyrosine phosphorylation on PKCδ and subsequent thromboxane generation through tyrosine kinase, Lyn, in platelets.

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Bitter stimuli induce Ca2+ signaling and CCK release in enteroendocrine STC-1 cells: role of L-type voltage-sensitive Ca2+ channels

AJP Cell Physiology ◽

10.1152/ajpcell.00003.2006 ◽

2006 ◽

Vol 291 (4) ◽

pp. C726-C739 ◽

Cited By ~ 141

Author(s):

Monica C. Chen ◽

S. Vincent Wu ◽

Joseph R. Reeve ◽

Enrique Rozengurt

Keyword(s):

Endocrine Cells ◽

Bitter Taste ◽

Dependent Manner ◽

Gi Tract ◽

Intracellular Ca ◽

Α Subunits ◽

Ca2 Channels ◽

Cck Release

We previously demonstrated the expression of bitter taste receptors of the type 2 family (T2R) and the α-subunits of the G protein gustducin (Gαgust) in the rodent gastrointestinal (GI) tract and in GI endocrine cells. In this study, we characterized mechanisms of Ca2+ fluxes induced by two distinct T2R ligands: denatonium benzoate (DB) and phenylthiocarbamide (PTC), in mouse enteroendocrine cell line STC-1. Both DB and PTC induced a marked increase in intracellular [Ca2+] ([Ca2+]i) in a dose- and time-dependent manner. Chelating extracellular Ca2+ with EGTA blocked the increase in [Ca2+]i induced by either DB or PTC but, in contrast, did not prevent the effect induced by bombesin. Thapsigargin blocked the transient increase in [Ca2+]i induced by bombesin, but did not attenuate the [Ca2+]i increase elicited by DB or PTC. These results indicate that Ca2+ influx mediates the increase in [Ca2+]i induced by DB and PTC in STC-1 cells. Preincubation with the L-type voltage-sensitive Ca2+ channel (L-type VSCC) blockers nitrendipine or diltiazem for 30 min inhibited the increase in [Ca2+]i elicited by DB or PTC. Furthermore, exposure to the L-type VSCCs opener BAY K 8644 potentiated the increase in [Ca2+]i induced by DB and PTC. Stimulation with DB also induced a marked increase in the release of cholecystokinin from STC-1 cells, an effect also abrogated by prior exposure to EGTA or L-type VSCC blockers. Collectively, our results demonstrate that bitter tastants increase [Ca2+]i and cholecystokinin release through Ca2+ influx mediated by the opening of L-type VSCCs in enteroendocrine STC-1 cells.

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Src Family Kinases in the Central Nervous System：Their Emerging Role in Pathophysiology of Migraine and Neuropathic Pain

Current Neuropharmacology ◽

10.2174/1570159x18666200814180218 ◽

2020 ◽

Vol 18 ◽

Author(s):

Lingdi Nie ◽

Wen-Rui Ye ◽

Shangbin Chen ◽

Domenico Chirchiglia ◽

Minyan Wang

Keyword(s):

Neuropathic Pain ◽

Tyrosine Kinases ◽

Neurological Diseases ◽

Src Family Kinases ◽

Signalling Pathways ◽

Pain Mechanisms ◽

Promising Therapeutic Target ◽

The Central Nervous System ◽

The Relationship

: Src family kinases (SFK) are a group of non-receptor tyrosine kinases which play a pivotal role in cellular responses and oncogenesis. Accumulating evidence suggest that SFK also act as a key component in signalling pathways of the central nervous system (CNS) in both physiological and pathological conditions. Despite the crucial role of SFK in signal transduction of the CNS, the relationship between SFK and molecules implicated in pain has been relatively unexplored. This article briefly reviews the recent advances uncovering the interplay of SFK with diverse membrane proteins and intracellular proteins in the CNS and the importance of SFK in the pathophysiology of migraine and neuropathic pain. Mechanisms underlying the role of SFK in these conditions and potential clinical applications of SFK inhibitors in neurological diseases are also summarised. We propose that SFK are the convergent point of signalling pathways in migraine and neuropathic pain and may constitute a promising therapeutic target for these diseases.

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Signalling by CGRP and Adrenomedullin in the Cerebellum and Other Systems

The Scientific World JOURNAL ◽

10.1100/tsw.2001.434 ◽

2001 ◽

Vol 1 ◽

pp. 11-11

Author(s):

David Poyner ◽

Heather Cater ◽

Nick Hartell ◽

Alex Conner ◽

Debbie Hay ◽

...

Keyword(s):

Tyrosine Kinases ◽

Transmitter Release ◽

Neurotransmitter Release ◽

Cultured Cells ◽

Signalling Pathways ◽

Nerve Terminals ◽

Parallel Fibre ◽

Isolated Tissues ◽

Stimulation Of

The best characterised signalling pathway activated by both CGRP and adrenomedullin is stimulation of adenylate cyclase via Gs. However, it is clear that in some circumstances the peptides can activate other signal transduction pathways, e.g., increases in intracellular calcium. Many of these signalling pathways can be observed in cultured cells but it is important also to examine isolated tissues to discover the full repertoire of transduction events. In the rat cerebellum there are receptors that respond to both CGRP and adrenomedullin. These seem to be located postsynaptically on Parallel Fibre nerve terminals and modulate transmission to Purkinje cells. Adrenomedullin acts via cAMP, apparently to augment neurotransmitter release. By contrast, CGRP decreases transmitter release, via a non-cAMP mediated pathway. We are currently examining the role of NO and tyrosine kinases in the responses to these peptides.

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Activation of Phosphatidylinositol-Linked Novel D1 Dopamine Receptors Inhibits High-Voltage-Activated Ca2+ Currents in Primary Cultured Striatal Neurons

Journal of Neurophysiology ◽

10.1152/jn.90345.2008 ◽

2009 ◽

Vol 101 (5) ◽

pp. 2230-2238 ◽

Cited By ~ 8

Author(s):

Li-Qun Ma ◽

Chao Liu ◽

Fang Wang ◽

Na Xie ◽

Jun Gu ◽

...

Keyword(s):

Dopamine Receptor ◽

High Voltage ◽

Dependent Manner ◽

Striatal Neurons ◽

Inhibitory Effects ◽

Patch Clamp Technique ◽

Whole Cell Patch Clamp ◽

Intracellular Ca ◽

Dose Dependent

Recent evidences indicate the existence of a putative novel phosphatidylinositol (PI)-linked D1 dopamine receptor that mediates excellent anti-Parkinsonian but less severe dyskinesia action. To further understand the basic physiological function of this receptor in brain, the effects of a PI-linked D1 dopamine receptor-selective agonist 6-chloro-7,8-dihydroxy-3-methyl-1-(3-methylphenyl)-2,3,4,5-tetrahydro-1H-3-benzazepine (SKF83959) on high-voltage activated (HVA) Ca2+ currents in primary cultured striatal neurons were investigated by whole cell patch-clamp technique. The results indicated that stimulation by SKF83959 induced an inhibition of HVA Ca2+ currents in a dose-dependent manner in substance-P (SP)-immunoreactive striatal neurons. Application of D1 receptor, but not D2, α1 adrenergic, 5-HT receptor, or cholinoceptor antagonist prevented SKF83959-induced reduction, indicating that a D1 receptor-mediated event assumed via PI-linked D1 receptor. SKF83959-induced inhibitory modulation was mediated by activation of phospholipase C (PLC), mobilization of intracellular Ca2+ stores and activation of calcineurin. Furthermore, the inhibitory effects were attenuated significantly by the L-type calcium channel antagonist nifedipine, suggesting that L-type calcium channels involved in the regulation induced by SKF83959. These findings may help to further understand the functional role of the PI-linked dopamine receptor in brain.

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Insulin secretion stimulated by l-arginine and its metabolite l-ornithine depends on Gαi2

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00337.2014 ◽

2014 ◽

Vol 307 (9) ◽

pp. E800-E812 ◽

Cited By ~ 15

Author(s):

Veronika Leiss ◽

Katarina Flockerzie ◽

Ana Novakovic ◽

Michaela Rath ◽

Annika Schönsiegel ◽

...

Keyword(s):

Insulin Secretion ◽

Amino Acid ◽

Metabolic Phenotype ◽

Β Cells ◽

Β Cell ◽

Glucose Challenge ◽

Intracellular Ca ◽

Plasma Insulin Levels

Bordetella pertussis toxin (PTx), also known as islet-activating protein, induces insulin secretion by ADP-ribosylation of inhibitory G proteins. PTx-induced insulin secretion may result either from inactivation of Gαo proteins or from combined inactivation of Gαo, Gαi1, Gαi2, and Gαi3 isoforms. However, the specific role of Gαi2 in pancreatic β-cells still remains unknown. In global (Gαi2−/−) and β-cell-specific (Gαi2βcko) gene-targeted Gαi2 mouse models, we studied glucose homeostasis and islet functions. Insulin secretion experiments and intracellular Ca2+ measurements were used to characterize Gαi2 function in vitro. Gαi2−/− and Gαi2βcko mice showed an unexpected metabolic phenotype, i.e., significantly lower plasma insulin levels upon intraperitoneal glucose challenge in Gαi2−/− and Gαi2βcko mice, whereas plasma glucose concentrations were unchanged in Gαi2−/− but significantly increased in Gαi2βcko mice. These findings indicate a novel albeit unexpected role for Gαi2 in the expression, turnover, and/or release of insulin from islets. Detection of insulin secretion in isolated islets did not show differences in response to high (16 mM) glucose concentrations between control and β-cell-specific Gαi2-deficient mice. In contrast, the two- to threefold increase in insulin secretion evoked by l-arginine or l-ornithine (in the presence of 16 mM glucose) was significantly reduced in islets lacking Gαi2. In accord with a reduced level of insulin secretion, intracellular calcium concentrations induced by the agonistic amino acid l-arginine did not reach control levels in β-cells. The presented analysis of gene-targeted mice provides novel insights in the role of β-cell Gαi2 showing that amino acid-induced insulin-release depends on Gαi2.

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Molecular Substrates Mediating Lanthanide-Evoked Neurotransmitter Release in Central Synapses

Journal of Neurophysiology ◽

10.1152/jn.90404.2008 ◽

2008 ◽

Vol 100 (4) ◽

pp. 2089-2100 ◽

Cited By ~ 8

Author(s):

ChiHye Chung ◽

Ferenc Deák ◽

Ege T. Kavalali

Keyword(s):

Neurotransmitter Release ◽

Surface Membrane ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Protein Receptor ◽

Rapid Action ◽

Intracellular Ca ◽

Synaptotagmin 1 ◽

Molecular Substrates ◽

Central Synapses

Noncanonical secretagogues such as hypertonicity or α-latrotoxin have been extremely informative in studying neurotransmission. Lanthanum and lanthanides can also trigger neurotransmitter release through an unknown mechanism. Here, we studied the effect of lanthanides on neurotransmission in hippocampal cultures. Application of 2 mM La3+ caused rapid and robust neurotransmitter release within seconds. In addition, transient application of La3+ uncovered a sustained facilitation of miniature neurotransmission. The response to La3+ was detectable at 2 μM and increased in a concentration-dependent manner ≤2 mM. Rapid effect of La3+ was independent of extracellular and intracellular Ca2+ and did not require La3+ entry into cells or activation of phospholipaseCβ. Synapses deficient in synaptobrevin-2, the major synaptic vesicle soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein in the brain, did not display any rapid release in response to La3+, whereas the slow facilitation of release detected after La3+ removal remained intact. In contrast, preincubation with intracellular Ca2+ chelators selectively attenuated the delayed release triggered by La3+. Moreover, synapses deficient in synaptotagmin-1 maintained a rapid response to La3+, suggesting that La3+-triggered neurotransmitter release does not require synaptotagmin-1 as a sensor. Therefore La3+ has two separate effects on synaptic transmission. For its rapid action, La3+ interacts with a target on the surface membrane, and unlike other forms of release, it triggers strictly synaptobrevin-2–dependent fusion, implying that in central synapses synaptobrevin-2 function is secretagogue specific. For the delayed action, La3+ may act intracellularly after its entry or through intracellular Ca2+ via a mechanism that does not require synaptobrevin-2.

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Association of p62, a multifunctional SH2- and SH3-domain-binding protein, with src family tyrosine kinases, Grb2, and phospholipase C gamma-1.

Molecular and Cellular Biology ◽

10.1128/mcb.15.1.186 ◽

1995 ◽

Vol 15 (1) ◽

pp. 186-197 ◽

Cited By ~ 135

Author(s):

S Richard ◽

D Yu ◽

K J Blumer ◽

D Hausladen ◽

M W Olszowy ◽

...

Keyword(s):

Phospholipase C ◽

Tyrosine Phosphorylation ◽

Tyrosine Kinases ◽

Sh3 Domain ◽

Sh2 Domain ◽

Src Family Kinases ◽

Src Family Kinase ◽

Sh3 Domains ◽

Sh2 Domains ◽

Src Family Tyrosine Kinases

src family tyrosine kinases contain two noncatalytic domains termed src homology 3 (SH3) and SH2 domains. Although several other signal transduction molecules also contain tandemly occurring SH3 and SH2 domains, the function of these closely spaced domains is not well understood. To identify the role of the SH3 domains of src family tyrosine kinases, we sought to identify proteins that interacted with this domain. By using the yeast two-hybrid system, we identified p62, a tyrosine-phosphorylated protein that associates with p21ras GTPase-activating protein, as a src family kinase SH3-domain-binding protein. Reconstitution of complexes containing p62 and the src family kinase p59fyn in HeLa cells demonstrated that complex formation resulted in tyrosine phosphorylation of p62 and was mediated by both the SH3 and SH2 domains of p59fyn. The phosphorylation of p62 by p59fyn required an intact SH3 domain, demonstrating that one function of the src family kinase SH3 domains is to bind and present certain substrates to the kinase. As p62 contains at least five SH3-domain-binding motifs and multiple tyrosine phosphorylation sites, p62 may interact with other signalling molecules via SH3 and SH2 domain interactions. Here we show that the SH3 and/or SH2 domains of the signalling proteins Grb2 and phospholipase C gamma-1 can interact with p62 both in vitro and in vivo. Thus, we propose that one function of the tandemly occurring SH3 and SH2 domains of src family kinases is to bind p62, a multifunctional SH3 and SH2 domain adapter protein, linking src family kinases to downstream effector and regulatory molecules.

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