COOH-terminal association of human smooth muscle calcium channel Cav1.2b with Src kinase protein binding domains: effect of nitrotyrosylation

The carboxyl terminus of the calcium channel plays an important role in the regulation of calcium entry, signal transduction, and gene expression. Potential protein-protein interaction sites within the COOH terminus of the L-type calcium channel include those for the SH3 and SH2 binding domains of c-Src kinase that regulates calcium currents in smooth muscle. In this study, we examined the binding sites involved in Src kinase-mediated phosphorylation of the human voltage-gated calcium channel (Cav) 1.2b (hCav1.2b) and the effect of nitrotyrosylation. Cotransfection of human embryonic kidney (HEK)-293 cells with hCav1.2b and c-Src resulted in tyrosine phosphorylation of the calcium channel, which was prevented by nitration of tyrosine residues by peroxynitrite. Whole cell calcium currents were reduced by 58 + 5% by the Src kinase inhibitor PP2 and 64 + 6% by peroxynitrite. Nitrotyrosylation prevented Src-mediated regulation of the currents. Glutathione S-transferase fusion protein of the distal COOH terminus of hCav1.2b (1809-2138) bound to SH2 domain of Src following tyrosine phosphorylation, while binding to SH3 required the presence of the proline-rich motif. Site-directed mutation of Y2134 prevented SH2 binding and resulted in reduced phosphorylation of hCav1.2b. Within the distal COOH terminus, single, double, or triple mutations of Y1837, Y1861, and Y2134 were constructed and expressed in HEK-293 cells. The inhibitory effects of PP2 and peroxynitrite on calcium currents were significantly reduced in the double mutant Y1837-2134F. These data demonstrate that the COOH terminus of hCav1.2b contains sites for the SH2 and SH3 binding of Src kinase. Nitrotyrosylation of these sites prevents Src kinase regulation and may be importantly involved in calcium influx regulation during inflammation.

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Phosphoinositide 3-kinase-dependent phosphorylation of the dual adaptor for phosphotyrosine and 3-phosphoinositides by the Src family of tyrosine kinase

Biochemical Journal ◽

10.1042/bj3490605 ◽

2000 ◽

Vol 349 (2) ◽

pp. 605-610 ◽

Cited By ~ 14

Author(s):

Simon DOWLER ◽

Leire MONTALVO ◽

Doreen CANTRELL ◽

Nick MORRICE ◽

Dario R. ALESSI

Keyword(s):

Growth Factor ◽

Tyrosine Kinase ◽

Active Form ◽

Adaptor Protein ◽

Sh2 Domain ◽

Ph Domain ◽

Hek 293 ◽

Hek 293 Cells ◽

293 Cells ◽

Phosphoinositide 3 Kinase

We recently identified a novel adaptor protein, termed dual adaptor for phosphotyrosine and 3-phosphoinositides (DAPP1), that possesses a Src homology (SH2) domain and a pleckstrin homology (PH) domain. DAPP1 exhibits a high-affinity interaction with PtdIns(3,4,5)P3 and PtdIns(3,4)P2, which bind to the PH domain. In the present study we show that when DAPP1 is expressed in HEK-293 cells, the agonists insulin, insulin-like growth factor-1 and epidermal growth factor induce the phosphorylation of DAPP1 at Tyr139. Treatment of cells with phosphoinositide 3-kinase (PI 3-kinase) inhibitors or expression of a dominant-negative PI 3-kinase prevent phosphorylation of DAPP1 at Tyr139, and a PH-domain mutant of DAPP1, which does not interact with PtdIns(3,4,5)P3 or PtdIns(3,4)P2, is not phosphorylated at Tyr139 following agonist stimulation of cells. Overexpression of a constitutively active form of PI 3-kinase induced the phosphorylation of DAPP1 in unstimulated cells. We demonstrated that Tyr139 of DAPP1 is likely to be phosphorylated in vivo by a Src-family tyrosine kinase, since the specific Src-family inhibitor, PP2, but not an inactive variant of this drug, PP3, prevented the agonist-induced tyrosine phosphorylation of DAPP1. Src, Lyn and Lck tyrosine kinases phosphorylate DAPP1 at Tyr139in vitro at similar rates in the presence or absence of PtdIns(3,4,5)P3, and overexpression of these kinases in HEK-293 cells induces the phosphorylation of Tyr139. These findings indicate that, following activation of PI 3-kinases, PtdIns(3,4,5)P3 or PtdIns(3,4)P2 bind to DAPP1, recruiting it to the plasma membrane where it becomes phosphorylated at Tyr139 by a Src-family tyrosine kinase.

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Functional Studies of RYR1 Mutations in the Skeletal Muscle Ryanodine Receptor Using Human RYR1 Complementary DNA

Anesthesiology ◽

10.1097/aln.0b013e3181d69283 ◽

2010 ◽

Vol 112 (6) ◽

pp. 1350-1354 ◽

Cited By ~ 20

Author(s):

Keisaku Sato ◽

Neil Pollock ◽

Kathryn M. Stowell

Keyword(s):

Skeletal Muscle ◽

Malignant Hyperthermia ◽

Calcium Channel ◽

Ryanodine Receptor ◽

Human Skeletal Muscle ◽

Binding Assays ◽

Complementary Dna ◽

Hek 293 ◽

Hek 293 Cells ◽

293 Cells

Background Malignant hyperthermia is associated with mutations within the gene encoding the skeletal muscle ryanodine receptor, the calcium channel that releases Ca from sarcoplasmic reticulum stores triggering muscle contraction, and other metabolic activities. More than 200 variants have been identified in the ryanodine receptor, but only some of these have been shown to functionally affect the calcium channel. To implement genetic testing for malignant hyperthermia, variants must be shown to alter the function of the channel. A number of different ex vivo methods can be used to demonstrate functionality, as long as cells from human patients can be obtained and cultured from at least two unrelated families. Because malignant hyperthermia is an uncommon disorder and many variants seem to be private, including the newly identified H4833Y mutation, these approaches are limited. Methods The authors cloned the human skeletal muscle ryanodine receptor complementary DNA and expressed both normal and mutated forms in HEK-293 cells and carried out functional analysis using ryanodine binding assays in the presence of a specific agonist, 4-chloro-m-cresol, and the antagonist Mg. Results Transiently expressed human ryanodine receptor proteins colocalized with an endoplasmic reticulum marker in HEK-293 cells. Ryanodine binding assays confirmed that mutations causing malignant hyperthermia resulted in a hypersensitive channel, while those causing central core disease resulted in a hyposensitive channel. Conclusions The functional assays validate recombinant human skeletal muscle ryanodine receptor for analysis of variants and add an additional mutation (H4833Y) to the repertoire of mutations that can be used for the genetic diagnosis of malignant hyperthermia.

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Pore properties and ionic block of the rabbit epithelial calcium channel expressed in HEK 293 cells

The Journal of Physiology ◽

10.1111/j.1469-7793.2001.0183l.x ◽

2001 ◽

Vol 530 (2) ◽

pp. 183-191 ◽

Cited By ~ 69

Author(s):

Rudi Vennekens ◽

Jean Prenen ◽

Joost G. J. Hoenderop ◽

René J. M. Bindels ◽

Guy Droogmans ◽

...

Keyword(s):

Calcium Channel ◽

Hek 293 ◽

Hek 293 Cells ◽

293 Cells ◽

Pore Properties

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Mutation of protein kinase C phosphorylation site S1076 on α-subunits affects BKCa channel activity in HEK-293 cells

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.90518.2008 ◽

2009 ◽

Vol 297 (4) ◽

pp. L758-L766 ◽

Cited By ~ 10

Author(s):

Shu Zhu ◽

Darren D. Browning ◽

Richard E. White ◽

David Fulton ◽

Scott A. Barman

Keyword(s):

Smooth Muscle ◽

Protein Kinase ◽

Phosphorylation Site ◽

Channel Activity ◽

Excitatory Effect ◽

Hek 293 ◽

Hek 293 Cells ◽

293 Cells ◽

Smooth Muscle Function ◽

Α Subunits

Large conductance, calcium- and voltage-activated potassium (BKCa) channels are important modulators of pulmonary vascular smooth muscle membrane potential, and phosphorylation of BKCa channels by protein kinases regulates pulmonary arterial smooth muscle function. However, little is known about the effect of phosphorylating specific channel subunits on BKCa channel activity. The present study was done to determine the effect of mutating protein kinase C (PKC) phosphorylation site serine 1076 (S1076) on transfected human BKCa channel α-subunits in human embryonic kidney (HEK-293) cells, a heterologous expression system devoid of endogenous BKCa channels. Results showed that mutating S1076 altered the effect of PKC activation on BKCa channels in HEK-293 cells. Specifically, the phospho-deficient mutation BKCa-α(S1076A)/β1 attenuated the excitatory effect of the PKC activator phorbol myristate acetate (PMA) on BKCa channels, whereas the phospho-mimetic mutation BKCa-α(S1076E)/β1 increased the excitatory effect of PMA on BKCa channels. In addition, the phospho-null mutation S1076A blocked the activating effect of cGMP-dependent protein kinase G (PKG) on BKCa channels. Collectively, these results suggest that specific putative PKC phosphorylation site(s) on human BKCa channel α-subunits influences BKCa channel activity, which may subsequently alter pulmonary vascular smooth muscle function and tone.

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Tyrosine-phosphorylation-dependent and Rho-protein-mediated control of cellular phosphatidylinositol 4,5-bisphosphate levels

Biochemical Journal ◽

10.1042/bj3340625 ◽

1998 ◽

Vol 334 (3) ◽

pp. 625-631 ◽

Cited By ~ 21

Author(s):

Ulrich RÜMENAPP ◽

Martina SCHMIDT ◽

Simone OLESCH ◽

Sabine OTT ◽

Christoph von EICHEL-STREIBER ◽

...

Keyword(s):

Tyrosine Kinase ◽

Tyrosine Phosphorylation ◽

Cellular Level ◽

Tyrosine Phosphatases ◽

Rho Proteins ◽

Toxin B ◽

Hek 293 ◽

Hek 293 Cells ◽

293 Cells ◽

Tyrphostin 23

The polyphosphoinositide PtdIns(4,5)P2, best known as a substrate for phospholipase C isozymes, has recently been recognized to be involved in a variety of other cellular processes. The aim of this study was to examine whether the cellular levels of this versatile phospholipid are controlled by tyrosine phosphorylation. The studies were performed in human embryonic kidney (HEK)-293 cells stably expressing the M3 muscarinic acetylcholine receptor. Inhibition of tyrosine phosphatases by pervanadate induced an up-to-approx.-2.5-fold increase in the total cellular level of PtdIns(4,5)P2, which was both time- and concentration-dependent. In contrast, the tyrosine kinase inhibitors, genistein and tyrphostin 23, caused a rapid and specific fall in the cellular PtdIns(4,5)P2 level and prevented the stimulatory effect of pervanadate on PtdIns(4,5)P2 formation. Inactivation of Rho proteins by Clostridium difficile toxin B caused a similar fall in the HEK-293 cell PtdIns(4,5)P2 level, which was not altered by additional genistein treatment. Furthermore, toxin B treatment abolished the pervanadate-induced increase in PtdIns(4,5)P2 levels. As PtdIns(4,5)P2 is an essential stimulatory cofactor for phospholipase D (PLD) enzymes, we finally examined the effects of the agents regulating PtdIns(4,5)P2 levels on PLD activity in HEK-293 cells. Inhibition of tyrosine phosphatases by pervanadate caused an increase in PLD activity, which was susceptible to genistein and tyrphostin 23, and which was abolished by prior treatment with toxin B. In conclusion, the data presented indicate that the cellular level of the multifunctional phospholipid, PtdIns(4,5)P2, in HEK-293 cells is controlled by a tyrosine-kinase-dependent mechanism and that this process apparently involves Rho proteins, as found similarly for tyrosine-phosphorylation-induced PLD activation.

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c-Src Regulates Clathrin Adapter Protein 2 Interaction with β-Arrestin and the Angiotensin II Type 1 Receptor during Clathrin- Mediated Internalization

Molecular Endocrinology ◽

10.1210/me.2004-0246 ◽

2005 ◽

Vol 19 (2) ◽

pp. 491-503 ◽

Cited By ~ 55

Author(s):

Delphine Fessart ◽

May Simaan ◽

Stéphane A. Laporte

Keyword(s):

Angiotensin Ii ◽

Src Kinase ◽

Receptor Internalization ◽

Ang Ii ◽

Coated Pits ◽

Hek 293 ◽

Hek 293 Cells ◽

293 Cells

Abstract β-Arrestins are multifunctional adapters involved in the internalization and signaling of G protein-coupled receptors (GPCRs). They target receptors to clathrin-coated pits (CCPs) through binding with clathrin and clathrin adapter 2 (AP-2) complex. They also act as transducers of signaling by recruiting c-Src kinase to certain GPCRs. Here we sought to determine whether c-Src regulates the recruitment of AP-2 to β-arrestin and the angiotensin II (Ang II) type 1 receptor (AT1R) during internalization. We show that the agonist stimulation of native AT1R in vascular smooth muscle cells (VSMCs) induces the formation of an endogenous complex containing c-Src, β-arrestins and AP-2. In vitro studies using coimmunoprecipitation experiments and a yeast three-hybrid assay reveal that c-Src stabilizes the agonist-independent association between β-arrestin2 and the β-subunit of AP-2 independently of the kinase activity of c-Src. However, although c-Src expression promoted the rapid dissociation of AP-2 from both β-arrestin and AT1R after receptor stimulation, a kinase-inactive mutant of c-Src failed to induce the dissociation of AP-2 from the agonist-occupied receptor. Thus, the consequence of c-Src in regulating the dissociation of AP-2 from the receptor was also examined on the internalization of AT1R by depleting c-Src in human embryonic kidney (HEK) 293 cells using a small interfering RNA strategy. Experiments in c-Src depleted cells reveal that AT1R remained mostly colocalized with AP-2 at the plasma membrane after Ang II stimulation, consistent with the observed delay in receptor internalization. Moreover, coimmunoprecipitation experiments in c-Src depleted HEK 293 cells and VSMCs showed an increased association of AP-2 to the agonist-occupied AT1R and β-arrestin, respectively. Together, our results support a role for c-Src in regulating the dissociation of AP-2 from agonist-occupied AT1R and β-arrestin during the clathrin-mediated internalization of receptors and suggest a novel function for c-Src kinase in the internalization of AT1R.

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Differential modulation of L-type calcium channel subunits by oleate

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.90237.2008 ◽

2008 ◽

Vol 294 (6) ◽

pp. E1178-E1186 ◽

Cited By ~ 14

Author(s):

Yingrao Tian ◽

Richard F. Corkey ◽

Gordon C. Yaney ◽

Paula B. Goforth ◽

Leslie S. Satin ◽

...

Keyword(s):

Fatty Acids ◽

Insulin Secretion ◽

Calcium Channel ◽

Dependent Manner ◽

Β Cells ◽

Β Cell ◽

Nonesterified Fatty Acids ◽

Hek 293 ◽

Hek 293 Cells ◽

293 Cells

Nonesterified fatty acids such as oleate and palmitate acutely potentiate insulin secretion from pancreatic islets in a glucose-dependent manner. In addition, recent studies show that fatty acids elevate intracellular free Ca2+ and increase voltage-gated Ca2+ current in mouse β-cells, although the mechanisms involved are poorly understood. Here we utilized a heterologous system to express subunit-defined voltage-dependent L-type Ca2+ channels (LTCC) and demonstrate that β-cell calcium may increase in part from an interaction between fatty acid and specific calcium channel subunits. Distinct functional LTCC were assembled in both COS-7 and HEK-293 cells by expressing either one of the EYFP-tagged L-type α1-subunits (β-cell Cav1.3 or lung Cav1.2) and ERFP-tagged islet β-subunits (iβ2a or iβ3). In COS-7 cells, elevations in intracellular Ca2+ mediated by LTCC were enhanced by an oleate-BSA complex. To extend these findings, Ca2+ current was measured in LTCC-expressing HEK-293 cells that revealed an increase in peak Ca2+ current within 2 min after addition of the oleate complex, with maximal potentiation occurring at voltages <0 mV. Both Cav1.3 and Cav1.2 were modulated by oleate, and the presence of different auxiliary β-subunits resulted in differential augmentation. The potentiating effect of oleate on Cav1.2 was abolished by the pretreatment of cells with triacsin C, suggesting that long-chain CoA synthesis is necessary for Ca2+ channel modulation. These results show for the first time that two L-type Ca2+ channels expressed in β-cells (Cav1.3 and Cav1.2) appear to be targeted by nonesterified fatty acids. This effect may account in part for the acute potentiation of glucose-dependent insulin secretion by fatty acids.

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Identification and Functional Characterization of Protein Kinase A-catalyzed Phosphorylation of Potassium Channel Kv1.2 at Serine 449

Journal of Biological Chemistry ◽

10.1074/jbc.m109.010918 ◽

2009 ◽

Vol 284 (24) ◽

pp. 16562-16574 ◽

Cited By ~ 19

Author(s):

Rosalyn P. Johnson ◽

Ahmed F. El-Yazbi ◽

Morgan F. Hughes ◽

David C. Schriemer ◽

Emma J. Walsh ◽

...

Keyword(s):

Smooth Muscle ◽

Protein Kinase ◽

Protein Kinase A ◽

Delayed Rectifier ◽

Hek 293 ◽

Hek 293 Cells ◽

Maldi Tof ◽

293 Cells ◽

Kinase A

Vascular smooth muscle Kv1 delayed rectifier K+ channels (KDR) containing Kv1.2 control membrane potential and thereby regulate contractility. Vasodilatory agonists acting via protein kinase A (PKA) enhance vascule smooth muscle Kv1 activity, but the molecular basis of this regulation is uncertain. We characterized the role of a C-terminal phosphorylation site, Ser-449, in Kv1.2 expressed in HEK 293 cells by biochemical and electrophysiological methods. We found that 1) in vitro phosphorylation of Kv1.2 occurred exclusively at serine residues, 2) one major phosphopeptide that co-migrated with 449pSASTISK was generated by proteolysis of in vitro phosphorylated Kv1.2, 3) the peptide 445KKSRSASTISK exhibited stoichiometric phosphorylation by PKA in vitro, 4) matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectroscopy (MS) and MS/MS confirmed in vitro Ser-449 phosphorylation by PKA, 5) in situ phosphorylation at Ser-449 was detected in HEK 293 cells by MALDI-TOF MS followed by MS/MS. MIDAS (multiple reaction monitoring-initiated detection and sequencing) analysis revealed additional phosphorylated residues, Ser-440 and Ser-441, 6) in vitro32P incorporation was significantly reduced in Kv1.2-S449A, Kv1.2-S449D, and Kv1.2-S440A/S441A/S449A mutant channels, but Kv1.2-S440A/S441A was identical to wild-type Kv1.2 (Kv1.2-WT), and 7) bath applied 8-Br-cAMP or dialysis with PKA catalytic subunit (cPKA) increased Kv1.2-WT but not Kv1.2-S449A current amplitude. cPKA increased Kv1.2-WT current in inside-out patches. Rp-CPT-cAMPS reduced Kv1.2-WT current, blocked the increase due to 8-Br-cAMP, but had no effect on Kv1.2-S449A. cPKA increased current due to double mutant Kv1.2-S440A/S441A but had no effect on Kv1.2-S449D or Kv1.2-S440A/S441A/S449A. We conclude that Ser-449 in Kv1.2 is a site of PKA phosphorylation and a potential molecular mechanism for Kv1-containing KDR channel modulation by agonists via PKA activation.

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Characteristics and requirements of basal autophagy in HEK 293 cells

Autophagy ◽

10.4161/auto.25455 ◽

2013 ◽

Vol 9 (9) ◽

pp. 1407-1417 ◽

Cited By ~ 41

Author(s):

Patience Musiwaro ◽

Matthew Smith ◽

Maria Manifava ◽

Simon A. Walker ◽

Nicholas T. Ktistakis

Keyword(s):

Hek 293 ◽

Hek 293 Cells ◽

293 Cells

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Halothane Inhibition of Recombinant Cardiac L-type Ca2+ Channels Expressed in HEK-293 Cells

Anesthesiology ◽

10.1097/00000542-200512000-00009 ◽

2005 ◽

Vol 103 (6) ◽

pp. 1156-1166 ◽

Cited By ~ 7

Author(s):

Kevin J. Gingrich ◽

Son Tran ◽

Igor M. Nikonorov ◽

Thomas J. Blanck

Keyword(s):

Charge Transfer ◽

Calcium Channels ◽

Dependent Manner ◽

Current Time ◽

Hek 293 ◽

Hek 293 Cells ◽

293 Cells ◽

Dependent Inactivation ◽

Voltage Dependent

Background Volatile anesthetics depress cardiac contractility, which involves inhibition of cardiac L-type calcium channels. To explore the role of voltage-dependent inactivation, the authors analyzed halothane effects on recombinant cardiac L-type calcium channels (alpha1Cbeta2a and alpha1Cbeta2aalpha2/delta1), which differ by the alpha2/delta1 subunit and consequently voltage-dependent inactivation. Methods HEK-293 cells were transiently cotransfected with complementary DNAs encoding alpha1C tagged with green fluorescent protein and beta2a, with and without alpha2/delta1. Halothane effects on macroscopic barium currents were recorded using patch clamp methodology from cells expressing alpha1Cbeta2a and alpha1Cbeta2aalpha2/delta1 as identified by fluorescence microscopy. Results Halothane inhibited peak current (I(peak)) and enhanced apparent inactivation (reported by end pulse current amplitude of 300-ms depolarizations [I300]) in a concentration-dependent manner in both channel types. alpha2/delta1 coexpression shifted relations leftward as reported by the 50% inhibitory concentration of I(peak) and I300/I(peak)for alpha1Cbeta2a (1.8 and 14.5 mm, respectively) and alpha1Cbeta2aalpha2/delta1 (0.74 and 1.36 mm, respectively). Halothane reduced transmembrane charge transfer primarily through I(peak) depression and not by enhancement of macroscopic inactivation for both channels. Conclusions The results indicate that phenotypic features arising from alpha2/delta1 coexpression play a key role in halothane inhibition of cardiac L-type calcium channels. These features included marked effects on I(peak) inhibition, which is the principal determinant of charge transfer reductions. I(peak) depression arises primarily from transitions to nonactivatable states at resting membrane potentials. The findings point to the importance of halothane interactions with states present at resting membrane potential and discount the role of inactivation apparent in current time courses in determining transmembrane charge transfer.

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